Human E3α ubiquitin ligase family

ABSTRACT

The present invention relates to a novel polypeptide encoding a protein which is the full length human ortholog of E3α ubiquitin ligase. The invention also relates to vector, host cells, antibodies and recombinant methods for producing the polypeptide. In addition, the invention discloses therapeutic, diagnostic and research utilities for these and related products.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.09/724,126, which was filed Nov. 28, 2000, now U.S. Pat. No. 6,706,505which in turn claims priority under 35 U.S.C. § 119 from U.S.provisional patent application Ser. No. 60/187,911, which was filed Mar.8, 2000, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention includes novel human E3α ubiquitin ligasepolypeptides (huE3αI and huE3αII) and nucleic acid molecules encodingthe same. The invention also relates to vectors, host cells, selectivebinding agents, such as antibodies, and methods for producing huE3αpolypeptides. Also provided for are methods for the diagnosis,treatment, amelioration and/or prevention of diseases associated withhuE3α polypeptides, as well as methods for identifying modulators ofhuE3α ligase activity.

BACKGROUND OF THE INVENTION

Technical advances in the identification, cloning, expression andmanipulation of nucleic acid molecules and deciphering of the humangenome have greatly accelerated the discovery of novel therapeuticsbased upon deciphering of the human genome. Rapid nucleic acidsequencing techniques can now generate sequence information atunprecedented rates and, coupled with computational analyses, allow theassembly of overlapping sequences into the partial and entire genomes aswell as the identification of polypeptide-encoding regions. A comparisonof a predicted amino acid sequence against a database compilation ofknown amino acid sequences can allow one to determine the extent ofhomology to previously identified sequences and/or structural landmarks.The cloning and expression of a polypeptide-encoding region of a nucleicacid molecule provides a polypeptide product for structural andfunctional analyses. The manipulation of nucleic acid molecules andencoded polypeptides to create variants and derivatives thereof mayconfer advantageous properties on a product for use as a therapeutic.

In spite of significant technical advances in genome research over thepast decade, the potential for the development of novel therapeuticsbased on the human genome is still largely unrealized. Many genesencoding potentially beneficial polypeptide therapeutics, or thoseencoding polypeptides which may act as “targets” for therapeuticmolecules, have still not been identified. In addition, structural andfunctional analyses of polypeptide products from many human genes havenot been undertaken.

Accordingly, it is an object of the invention to identify novelpolypeptides and nucleic acid molecules encoding the same which havediagnostic or therapeutic benefit.

Most types of intracellular proteins are degraded through theubiquitin-proteosome pathway. In this system, proteins are marked forprotesomal degradation by the conjugation of ubiquitin molecules to theprotein. Conjugation of the ubiquitin molecule initially involvesactivation by the E1 enzyme. Upon activation the ubiquitin molecule istransferred to the E2 enzyme which serves as a carrier-protein. The E2enzyme interacts with a specific E3 ligase family member. The E3 ligasebinds to proteins targeted for degradation and catalyzes the transfer ofubiquitin from the E2 carrier enzyme to the target protein. Since thetarget protein binds to the ligase prior to conjugatin, E3 ligase is therate limiting step for ubiquitin conjugation and determines thespecificity of the system. The ubiquitin chain serves as a degradationmarker for the 26S proteosome (See Ciechanover, EMBO J., 17: 7151–7160,1998).

There are only a few known E3 ligases and the sequence homology betweenthem is low. The E3α family is the main family of intracellularubiquitin ligases and is involved in N-end rule pathway of proteindegradation. The N-end rule states that there is a strong relationbetween the in vivo half-life of a protein and the identity of itsN-terminal amino acids. Accordingly, E3α enzyme binds directly to theprimary destabilizing N-terminal amino acid and catalyzes ubiquitinconjugation thereby targeting the protein for degradation. E3α familymembers also recognize non-N-end rule substrates (See Ciechanover, EMBOJ., 17: 7151–7160, 1998).

The E3α enzyme family currently consists of intracellular enzymesisolated from rabbit (Reiss and Hershiko, J. Biol. Chem. 265: 3685–3690,1990), mouse (Kwon et al., Proc. Natl. Acad. Sci., U.S.A 95: 7898–7903,1999), yeast (Bartel et al., EMBO J., 9: 3179–3189, 1990) and the C.elegans (Wilson et al., Nature, 368: 32–38, 1994; Genebank Accession No.U88308) counterparts termed UBR-1. Comparison of these known sequencesindicates regions of high similarity regions (I–V) which suggest theexistence of a distinct family. The regions of similarity containessential residues for the recognition of N-end rule substrates. Inregion I, the residues Cys-145, Val-146, Gly-173, and Asp-176 are knownto be necessary for type-1 substrate binding in yeast and are conservedin the mouse. In regions II and III, residues Asp-318, His-321, andGlu-560 are essential for type-2 substrate binding in yeast and are alsoconserved in the mouse. In addition, there is a conserved zinc-fingerdomain in region I and a conserved RING-H2 domain in region IV (Kwon etal., Proc. Natl. Acad. Sci., U.S.A, 95: 7898–7903, 1999).

The full length mouse E3α cDNA sequence and a partial human E3αnucleotide sequence (≈1 kb) have recently been cloned and characterizedas described in U.S. Pat. No. 5,861,312 and Kwon et al. (Proc. Natl.Acad. U.S.A., 95: 7898–7908, 1999). The full length mouse E3α cDNAsequence is 5271 bp in length and encodes a 1757 amino acid polypeptide.The mouse E3α gene is localized to the central region of chromosome 2and is highly expressed in skeletal muscle, heart and brain. The partialhuman E3α sequence was used to characterize tissue expression andchromosomal localization. This analysis indicated that the human E3αgene is located on chromosome 15q and exhibits a similar expressionpattern as mouse E3α with high expression in skeletal muscle, heart andbrain. As described herein, the present invention discloses two novel,full length, human E3α sequences (huE3αI and huE3αII) and a novel, fulllength mouse E3α sequence (muE3αII). Expression of huE3αI and huE3αIImRNA is highly enriched in skeletal muscle tissues. Functionally, huE3αpolypeptides are intracellular enzymes that control protein conjugationand degradation.

Increased proteolysis through the ubiquitin-proteosome pathway has beendetermined to be a major cause of rapid muscle wasting in manypathological states including but not limited to fasting, metabolicacidosis, muscle denervation, kidney failure, renal cachexia, uremia,diabetes mellitus, sepsis, AIDS wasting syndrome, cancer cachexia,negative nitrogen balance cachexia, burns and Cushing's syndrome (SeeMitch and Goldberg, New England J. Med, 335: 1897–1905, 1996). Studiesin animal models have shown that muscle wasting disorders are associatedwith increased ubiquitin content in muscles, increased levels of mRNAtranscripts encoding ubiquitin, E2 enzyme and proteosome subunit mRNA,and increased ubiquitin-conjugation to muscle-proteins (See Lecker etal., J. Nutr., 129: 227S–237S, 1999). In this context, the N-end rulepathway has been shown to play a role in muscle atrophy. E3α inhibitors,such as dipepetides and methyl ester, reduce the level of ubiquitinconjugation in atrophying rat muscles caused by sepsis, fasting andcancer cachexia (Soloman et al., Proc. Natl. Acad. Sci. U.S.A. 95:12602–12607, 1999). These observations indicate that E3α plays a role inthe overall increase in ubiquitination that is associated with and maymediate muscle atrophy in catabolic and other disease states.

Thus, identification of members of the N-end rule protein degradationpathway has led to a better understanding of protein degradation inhuman cells and the mechanisms of protein degradation in pathologicalcondition which involve muscle atrophy. Identification of the two novelhuman E3α ubiquitin ligase genes and polypeptides, as described herein,will further clarify the understanding of these processes and facilitatethe development of therapies for pathological conditions which involveabnormal or excessive protein degradation including conditions whichinvolve atrophy of muscle.

SUMMARY OF THE INVENTION

The present invention relates to novel human E3α nucleic acid moleculesand polypeptides encoded by these nucleic acid molecules.

The invention provides isolated nucleic acid molecules comprising orconsisting of a nucleotide sequence selected from the group consistingof:

-   -   a) the nucleotide sequence as set forth in SEQ ID NOS: 1 or 3;    -   b) a nucleotide sequence encoding the polypeptide set forth in        SEQ ID NOS: 2 and 4;    -   c) a nucleotide sequence which hybridizes under moderate or        highly stringent conditions to the compliments of (a) or (b);        and    -   d) a nucleotide complementary to (a)–(c)

The invention also provides isolated nucleic acid molecules comprising anucleotide sequence selected from the group consisting of:

-   -   a) a nucleotide sequence encoding a polypeptide that is at least        about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent        identical to the polypeptide set forth in SEQ ID NOS: 2 or 4,        wherein the polypeptide has an activity of the polypeptide set        forth in SEQ ID NOS: 2 or 4 and the percent identity for these        nucleic acid sequences are determined using a computer program        selected from the group consisting of GAP, BLASTP, BLASTN,        FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman        algorithm;    -   b) a nucleotide sequence encoding an allelic variant or splice        variant of the nucleotide sequence as set forth in SEQ ID NOS: 1        or 3;    -   c) the nucleotide sequence of the DNA insert in ATCC Deposit No.        PTA-1489 or PTA-1490;    -   d) a nucleotide sequence of SEQ ID NOS: 1; 3; (a); or (b)        encoding a polypeptide fragment of at least about 25 amino acid        residues, wherein the polypeptide has an activity of the        polypeptide set forth in SEQ ID NOS: 2 or 4;    -   e) a nucleotide sequence of SEQ ID NOS: 1, 3, or (a)–(c)        comprising a fragment of at least about 16 nucleotides;    -   f) a nucleotide sequence which hybridizes under moderately or        highly stringent conditions to the complement of any of (a)–(e);        and    -   g) a nucleotide sequence complementary to any of (a)–(d).

The invention also provides isolated nucleic acid molecules comprising anucleotide sequence selected from the group consisting of:

-   -   a) a nucleotide sequence encoding a polypeptide set forth in SEQ        ID NOS: 2 or 4 with at least one conservative amino acid        substitution, wherein the polypeptide has an activity of the        polypeptide set forth in SEQ ID NOS: 2 or 4;    -   b) a nucleotide sequence encoding a polypeptide set forth in SEQ        ID NOS: 2 or 4 with at least one amino acid insertion, wherein        the polypeptide has an activity of the polypeptide set forth in        SEQ ID NOS: 2 or 4;    -   c) a nucleotide sequence encoding a polypeptide set forth in SEQ        ID NOS: 2 or 4 with at least one amino acid deletion, wherein        the polypeptide has an activity of the polypeptide set forth in        SEQ ID NOS: 2 or 4;    -   d) a nucleotide sequence encoding a polypeptide set forth in SEQ        ID NOS: 2 or 4 which has a C- and/or N-terminal truncation,        wherein the polypeptide has an activity of the polypeptide set        forth in SEQ ID NOS: 2 or 4;    -   e) a nucleotide sequence encoding a polypeptide set forth in SEQ        ID NOS: 2 or 4 with at least one modification selected from the        group consisting of amino acid substitutions, amino acid        insertions, amino acid deletions, C-terminal truncation, and        N-terminal truncation, wherein the polypeptide has an activity        of the polypeptide set forth in SEQ ID NOS: 2 or 4;    -   f) a nucleotide sequence of (a)–(e) comprising a fragment of at        least about 16 nucleotides;    -   g) a nucleotide sequence which hybridizes under moderately or        highly stringent conditions to the complement of any of (a)–(f);        and    -   h) a nucleotide sequence complementary to any of (a)–(e).

The invention also provides isolated polypeptides comprising the aminoacid sequence selected from the group consisting of:

-   -   a) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4;    -   b) the mature amino acid sequence as set forth in SEQ ID NOS: 2        or 4 comprising a mature amino terminus at residues 1, and        optionally further comprising an amino terminal methionine;    -   c) an amino acid sequence that is at least about 70, 75, 80, 85,        90, 95, 96, 97, 98, or 99 percent identical to the amino acid        sequence of the polypeptide of SEQ ID NOS: 2 or 4 wherein the        polypeptide has an activity of the polypeptide set forth in SEQ        ID NOS: 2 or 4 and the percent identity for these amino acid        sequences are determined using a computer program selected from        the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA,        BLASTX, BestFit, and the Smith-Waterman algorithm.    -   d) a fragment of the amino acid sequence set forth in SEQ ID        NOS: 2 or 4 comprising at least about 25, 50, 75, 100, or        greater than 100 amino acid residues, wherein the fragment has        an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;    -   e) the amino acid sequence encoded by the DNA insert of ATCC        Deposit No. PTA-1489 or PTA-1490;    -   f) an amino acid sequence for an ortholog of SEQ ID NOS: 2 or 4;        including the murine ortholog set out as SEQ ID NO: 6.    -   g) an allelic variant or splice variant of (a), (b), (e) or (f);

The present invention also provides isolated polypeptides comprising theamino acid sequence selected from the group consisting of:

-   -   a) the amino acid as sequence set forth in SEQ ID NOS: 2 or 4        with at least one conservative amino acid substitution, wherein        the polypeptide has an activity of the polypeptide set forth in        SEQ ID NOS: 2 or 4;    -   b) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4        with at least one amino acid insertion, wherein the polypeptide        has an activity of the polypeptide set forth in SEQ ID NOS: 2 or        4;    -   c) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4        with at least one amino acid deletion, wherein the polypeptide        has an activity of the polypeptide set forth in SEQ ID NOS: 2 or        4;    -   d) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4        which has a C- and/or N-terminal truncation, wherein the        polypeptide has an activity of the polypeptide set forth in SEQ        ID NOS: 2 or 4; and    -   e) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4,        with at least one modification selected from the group        consisting of amino acid substitutions, amino acid insertions,        amino acid deletions, C-terminal truncation, and N-terminal        truncation, wherein the polypeptide has an activity of the        polypeptide set forth in SEQ ID NOS: 2 or 4.

The present invention provides expression vectors comprising the nucleicacid molecules set forth herein, host cells comprising the expressionvectors of the invention, and a method of producing a human E3αpolypeptide comprising culturing the host cells and optionally isolatingthe polypeptide so produced. An another embodiment provides for viralvectors comprising the nucleic acid molecules of the inventions. Furtherprovided is a process for determining whether a compound inhibits huE3αpolypeptide activity or production comprising exposing a host cellexpressing huE3α polypeptide to the compound, and measuring huE3αpolypeptide activity or production in said cell.

A transgenic non-human animal comprising a nucleic acid moleculeencoding a huE3α polypeptide is also encompassed by the invention. ThehuE3α nucleic acid molecules are introduced into the animal in a mannerthat allows expression and increased levels of a huE3α polypeptide,which may include increased circulating levels. The transgenic non-humananimal is preferably a mammal, and more preferably a rodent, such as arat or a mouse.

Also provided are derivatives of the huE3α polypeptides of the presentinvention, fusion polypeptides comprising the huE3α polypeptides of theinvention, and selective binding agents such as antibodies capable ofspecifically binding the polypeptides of the invention.

Pharmaceutical compositions comprising the nucleotides, polypeptides, orselective binding agents of the present invention and a carrier,adjuvant, solubilizer, stabilizer, anti-oxidant, or otherpharmaceutically acceptable formulation agent are also encompassed bythe invention. The pharmaceutical compositions include therapeuticallyeffective amounts of the nucleotides or polypeptides of the presentinvention, and involve methods of using the polypeptides and nucleicacid molecules.

The huE3α polypeptides and nucleic acid molecules of the presentinvention may be used for therapeutic or diagnostic purposes to treat,prevent, and/or detect diseases or disorders, including those recitedherein.

Methods of regulating expression and modulating (i.e., increasing ordecreasing) levels of a huE3α polypeptide are also encompassed by theinvention. One method comprises administering to an animal a nucleicacid molecule encoding a huE3α polypeptide. In another method, a nucleicacid molecule comprising elements that regulate or modulate theexpression of a huE3α polypeptide may be administered. Examples of thesemethods include gene therapy, cell therapy and antisense therapy asfurther described herein. Further provided is a method of identifying acompound which binds to a huE3α polypeptide comprising.

A device, comprising a membrane suitable for implantation and host cellsexpressing a huE3α polypeptide encapsulated within said membrane,wherein said membrane is permeable to said protein product andimpermeable to materials detrimental to said cells is also encompassedby the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A–1L shows the alignment of the amino acid sequences for huE3αI,huE3αII, muE3αI and muE3αII (SEQ ID NOS: 2, 4, 15 and 6, respectively).

FIG. 2 shows the results of a human multiple tissue Northern blotdetecting huE3αII expression.

FIG. 3 shows the results of a human multiple tissue Northern blotdetecting huE3αI expression.

FIG. 4 shows that transfection of 293T cells with huE3αI and huE3II cDNAstimulates the ubiquitination of endogenous proteins and exogenouslyadded α-lactalbumin in cell lysates. The left panel shows the results ofgel-shift assays of ubiquitinated proteins. The high molecular weightbands (above 18 kDa for endogenous proteins and above 33 kDa forα-lactalbumin) are identified as “¹²⁵I-Ubiquitin-protein conjugates”.The left panel plots the quantitative measurement of ubiquinatedproteins measured by a PhosphoImager.

FIG. 5 shows that transfection of C₂C₁₂ and L6 myotube cells with huE3αIand huE3II cDNA stimulates the ubiquitination of endogenous proteinscell lysates. The left panel shows the ubiquitinated high molecularweight bands (above 18 kDa for endogenous proteins) as“¹²⁵I-Ubiquitin-protein conjugates”. The left panel plots thequantitative measurement of ubiquinated proteins measured by aPhosphoImager.

FIG. 6 shows the ¹²⁵I-ubiquitin conjugation to endogenous muscleproteins and its sensitivity to selective inhibitors of E3α in muscleextracts from control and YAH-tumor bearing rats. Gel-shift assays ofmuscle extracts from control and tumor-bearing rats revealed theubiquitinated high molecular weight bands (above 18 kDa) denoted as“¹²⁵I-Ubiquitin-protein conjugates”. The left panel is muscle extractscollected 3 days post-implantation and the right panel is muscleextracts collected 5 days post-implantation.

FIG. 7 shows the ubiquitin conjugation to ¹²⁵I-α-lactalbumin in extractsfrom atrophying muscles in YAH-tumor bearing rats as western blots ofmuscle extracts from control and tumor-bearing rats with theubiquitinated high molecular weight bands (above 33 kDa) as“¹²⁵I-Lactalbumin-ubiquitin conjugation”. The left panel is muscleextracts collected 3 days post-implantation and the right panel ismuscle extracts collected 5 days post-implantation.

FIG. 8 shows Northern blot analysis of E3αI and E3αII expression inskeletal muscle in YAH-130 experimental cachexia model. The RNAexpression from pair-fed control rats and tumor-bearing rats werecompared 3 days (3 d) and 5 days (5 d) post-implantation.

FIG. 9 shows Northern blot analysis of E3αI and E3αII expression inskeletal (gastrocnemius) muscle and cardiac muscle in the C26experimental cacheixia model. The RNA expression from pair-fed controlrats and tumor-bearing mice were compared 12 days (12 d) and 17 days (17d) post-implantation.

FIG. 10 shows induction of E3αII expression by proinflammatory cytokinesTNFα and IL-6 in C₂C₁₂ myotube cultures on Northern blots. The RNAlevels of E3αII (upper panel) and E3αI (lower panel) were detected 3 or5 days after treatment with TNFα (left panel) and IL-6 (right panel).

FIG. 11 shows that IL-6 treatment causes a time-dependent accelerationof ubiquitination in differentiated C₂C₁₂ cells. This data exhibits theresults of a gel-shift assay showing the ubiquitinated high molecularweight bands denoted as “¹²⁵I-ubiquitin protein conjugates” (left panel)and is quantitated by a PhosphoImager in the right panel.

FIG. 12 shows that TNFα treatment causes a does-dependent accelerationof ubiquitination in differentiated C₂C₁₂ cells. This data is displayedas gel-shift assay results with the ubiquitinated high molecular weightbands denoted as “¹²⁵I-ubiquitin protein conjugates” (left panel) and isquantitated by a PhosphoImager in the right panel.

DETAILED DESCRIPTION OF THE INVENTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter describedtherein. All references cited in this application are expresslyincorporated by reference herein.

Definitions

The term “huE3α” encompasses two novel orthologs of human E3α ubiquitinligase described herein including huE3αI polynucleotide and polypeptide(SEQ ID NOS: 1 and 2, respectively) and huE3αII polynucleotide andpolypeptide (SEQ ID NOS: 3 and 4, respectively).

The term “huE3α nucleic acid molecule” or “polynucleotide” refers to anucleic acid molecules including a nucleotide sequence as set forth inSEQ ID NOS: 1 or 3, a nucleotide sequence encoding the polypeptide setforth in SEQ ID NOS: 2 or 4, a nucleotide sequence of the DNA insert inATCC deposit nos. PTA-1489 or PTA-1490, or nucleic acid molecule relatedthereto. Related nucleic acid molecules include a nucleotide sequencethat is at least about 70 percent identical to the nucleotide sequenceas shown in SEQ ID NOS: 1 or 3, or comprise or consist essentially of anucleotide sequence encoding a polypeptide that is at least about 70percent identical to the polypeptide set forth in SEQ ID NOS: 2 or 4. Inpreferred embodiments, these nucleotide sequences are about 75 percent,or about 80 percent, or about 85 percent, or about 90 percent, or about95, 96, 97, 98, or 99 percent identical to the nucleotide sequence asshown in SEQ ID NOS: 1 or 3, or the nucleotide sequences encode apolypeptide that is about 75 percent, or about 80 percent, or about 85percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percentidentical to the polypeptide sequence as set forth in SEQ ID NOS: 2 or4.

Related nucleic acid molecules also include fragments of the huE3αI orhu E3αII nucleic acid molecules which fragments contain at least about10 contiguous nucleotides, or about 15, or about 20, or about 25, orabout 50, or about 75, or about 100, or greater than about 100contiguous nucleotides of a huE3α nucleic acid molecule of SEQ ID NOS: 1or 3. Related nucleic acid molecules also include fragments of the abovehuE3α nucleic acid molecules which encode a polypeptide of at leastabout 25 amino acid residues, or about 50, or about 75, or about 100, orgreater than about 100 amino acid residues of the huE3α polypeptide ofSEQ ID NOS: 2 or 4. Related nucleic acid molecules also include anucleotide sequence encoding a polypeptide comprising or consistingessentially of a substitution, modification, addition and/or a deletionof one or more amino acid residues compared to the polypeptide set forthin SEQ ID NOS: 2 or 4. In addition, related huE3α nucleic acid moleculesinclude those molecules which comprise nucleotide sequences whichhybridize under moderately or highly stringent conditions as definedherein with the fully complementary sequence of any of the huE3α nucleicacid molecules of SEQ ID NOS: 1 or 3.

In preferred embodiments, the related nucleic acid molecules comprisesequences which hybridize under moderately or highly stringentconditions with a molecule having a sequence as shown in SEQ ID NOS: 1or 3, or of a molecule encoding a polypeptide, which polypeptidecomprises the sequence as shown in SEQ ID NOS: 2 or 4, or of a nucleicacid fragment as defined herein, or of a nucleic acid fragment encodinga polypeptide as defined herein or the complement of any or the forgoingmolecules. It is also understood that related nucleic acid moleculesinclude allelic or splice variants of a huE3α nucleic acid molecule ofSEQ ID NOS: 1 or 3, and include sequences which are complementary to anyof the above nucleotide sequences. The related encoded polypeptidespossess at least one activity of the polypeptide depicted in SEQ ID NOS:2 or 4.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that is free from at least one contaminatingnucleic acid molecule with which it is naturally associated. Preferably,the isolated nucleic acid molecule of the present invention issubstantially free from any other contaminating mammalian nucleic acidmolecule(s) which would interfere with its use in polypeptide productionor its therapeutic, diagnostic, or preventative use.

A “nucleic acid sequence” or “nucleic acid molecule” as used hereinrefer to a DNA or RNA sequence. The terms encompasses molecules formedfrom any of the known base analogs of DNA and RNA such as, but notlimited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylamino-methyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “operably linked” is used as recognized in the art to refer toan arrangement of flanking sequences wherein the flanking sequences sodescribed are configured or assembled so as to perform their usualfunction. Thus, a flanking sequence operably linked to a coding sequencemay be capable of effecting the replication, transcription and/ortranslation of the coding sequence. For example, a coding sequence isoperably linked to a promoter when the promoter is capable of directingtranscription of that coding sequence. A flanking sequence need not becontiguous with the coding sequence, so long as it functions correctly.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence andthe promoter sequence can still be considered “operably linked” to thecoding sequence.

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refer to one or more formulationmaterials suitable for accomplishing or enhancing delivery of the huE3αpolypeptide, huE3α nucleic acid molecule, or huE3α selective bindingagent as a pharmaceutical composition.

The term “allelic variant” refers to one of several possible naturallyoccurring alternate forms of a gene occupying a given locus on achromosome of an organism or a population of organisms.

The term “splice variant” refers to a nucleic acid molecule, usuallyRNA, which is generated by alternative processing of intron sequences inan RNA transcript of huE3α polypeptide amino acid sequence.

The term “expression vector” refers to a vector which is suitable fortransformation of a host cell and contains nucleic acid sequences whichdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “vector” is used as recognized in the art to refer to anymolecule (e.g., nucleic acid, plasmid, or virus) used to transfer codinginformation to a host cell.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, may be maintained transiently as an episomal element without beingreplicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, for example, Graham et al., Virology, 52: 456, 1973;Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratories, New York, 1989; Davis et al., Basic Methods inMolecular Biology, Elsevier, 1986; and Chu et al., Gene, 13: 197, 1981.Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed, by a vector bearing aselected gene of interest which is then expressed by the cell. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

The term “highly stringent conditions” refers to those conditions thatare designed to permit hybridization of DNA strands whose sequences arehighly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65–68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate,and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory,(Cold Spring Harbor, N.Y. 1989); Anderson et al., Nucleic AcidHybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford,England).

More stringent conditions (such as higher temperature, lower ionicstrength, higher formamide, or other denaturing agent) may also be used,however, the rate of hybridization will be affected. Other agents may beincluded in the hybridization and washing buffers for the purpose ofreducing non-specific and/or background hybridization. Examples are 0.1%bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodiumpyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO₄, or SDS, ficoll,Denhardt's solution, sonicated salmon sperm DNA (or othernon-complementary DNA), and dextran sulfate, although another suitableagents can also be used. The concentration and types of these additivescan be changed without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8–7.4, however, at typical ionic strength conditions, therate of hybridization is nearly independent of pH. (See Anderson et al.,Nucleic Acid Hybridisation: a Practical Approach, Ch. 4, IRL PressLimited (Oxford, England)).

Factors affecting the stability of DNA duplex include base composition,length, and degree of base pair mismatch. Hybridization conditions canbe adjusted by one skilled in the art in order to accommodate thesevariables and allow DNAs of different sequence relatedness to formhybrids. The melting temperature of a perfectly matched DNA duplex canbe estimated by the following equation:T _(m)(° C.)=81.5+16.6(log[Na+])+0.41(% G+C)−600/N−0.72(% formamide)where N is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

The term “moderately stringent conditions” refers to conditions underwhich a DNA duplex with a greater degree of base pair mismatching thancould occur tinder “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.01 5M sodiumchloride, 0.0015 M sodium citrate at 50–65° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 20% formamide at 37–50° C. By wayof example, a “moderately stringent” condition of 50° C. in 0.015 Msodium ion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is noabsolute distinction between “highly” and “moderately” stringentconditions. For example, at 0.015M sodium ion (no formamide), themelting temperature of perfectly matched long DNA is about 71° C. With awash at 65° C. (at the same ionic strength), this would allow forapproximately a 6% mismatch. To capture more distantly relatedsequences, one skilled in the art can simply lower the temperature orraise the ionic strength.

A good estimate of the melting temperature in 1 M NaCl* foroligonucleotide probes up to about 20 nt is given by:Tm=2° C. per A−T base pair+4° C. per G−C base pair*The sodium ion concentration in 6× salt sodium citrate (SSC) is 1M. SeeSuggs et al., Developmental Biology Using Purified Genes, p. 683, Brownand Fox (eds.) (1981).

High stringency washing conditions for oligonucleotides are usually at atemperature of 0–5° C. below the Tm of the oligonucleotide in 6×SSC,0.1% SDS for longer nucleotides.

The term “huE3α polypeptide” refers to a polypeptide comprising theamino acid sequence of huE3αI or huE3αII (SEQ ID NOS: 2 or 4,respectively), and related polypeptides having a natural sequence ormutated sequence. Related polypeptides include: allelic variants; splicevariants; fragments; derivatives; substitution, deletion, and insertionvariants; fusion polypeptides; and orthologs of the huE3α polypeptidesof SEQ ID NOS: 2 or 4, which possess at least one activity of thepolypeptide depicted in SEQ ID NOS: 2 or 4. Human E3α polypeptides maybe mature polypeptides, as defined herein, and may or may not have anamino terminal methionine residue, depending on the method by which theyare prepared.

The term “huE3α polypeptide fragment” refers to a polypeptide thatcomprises less than the full length amino acid sequence of a huE3αI orhuE3αII polypeptide set forth in SEQ ID NOS: 2 or 4, respectively. SuchhuE3α fragments can be 6 amino acids or more in length, and may arise,for example, from a truncation at the amino terminus (with or without aleader sequence), a truncation at the carboxy terminus, and/or aninternal deletion of one or more residues from the amino acid sequence.Human E3α fragments may result from alternative RNA splicing or from invivo protease activity. Membrane-bound forms of huE3α are alsocontemplated by the present invention. In preferred embodiments,truncations and/or deletions comprise about 10 amino acids, or about 20amino acids, or about 50 amino acids, or about 75 amino acids, or about100 amino acids, or more than about 100 amino acids. The polypeptidefragments so produced will comprise about 25 contiguous amino acids, orabout 50 amino acids, or about 75 amino acids, or about 100 amino acids,or about 150 amino acids, or about 200 amino acids. Such huE3αpolypeptide fragments may optionally comprise an amino terminalmethionine residue. It will be appreciated that such fragments can alsobe used, for example, to generate antibodies to huE3α polypeptides.

The term “huE3α polypeptide variants” refers to huE3α polypeptides whichcontain one or more amino acid sequence substitutions, deletions, and/oradditions as compared to the huE3α polypeptide amino acid sequence setforth as huE3αI or huE3αII (SEQ ID NOS: 2 or 4, respectively). Variantsmay be naturally occurring or artificially constructed. Such huE3αpolypeptide variants may be prepared from the corresponding nucleic acidmolecules encoding said variants, which have a DNA sequence that variesaccordingly from the DNA sequences for wild type huE3α polypeptides asset forth in SEQ ID NOS: 1 or 3. In preferred embodiments, the variantshave from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 20, orfrom 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, ormore than 100 amino acid substitutions, insertions, additions and/ordeletions, wherein the substitutions may be conservative, ornon-conservative, or any combination thereof.

One skilled in the art will be able to determine suitable variants ofthe native huE3α polypeptide using well known techniques. For example,one may predict suitable areas of the molecule that may be changedwithout destroying biological activity. Also, one skilled in the artwill realize that even areas that may be important for biologicalactivity or for structure may be subject to conservative amino acidsubstitutions without destroying the biological activity or withoutadversely affecting the polypeptide structure.

For predicting suitable areas of the molecule that may be changedwithout destroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of huE3α polypeptide to such similar polypeptides. After makingsuch a comparison, one skilled in the art can determine residues andportions of the molecules that are conserved among similar polypeptides.One skilled in the art would know that changes in areas of the huE3αmolecule that are not conserved would be less likely to adversely affectthe biological activity and/or structure of a huE3α polypeptide. Oneskilled in the art would also know that, even in relatively conservedregions, one may substitute chemically similar amino acids for thenaturally occurring residues while retaining activity (conservativeamino acid residue substitutions).

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one skilled inthe art can predict the importance of amino acid residues in a huE3αpolypeptide that correspond to amino acid residues that are importantfor activity or structure in similar polypeptides. One skilled in theart may opt for chemically similar amino acid substitutions for suchpredicted important amino acid residues of huE3α polypeptides.

If available, one skilled in the art can also analyze thethree-dimensional structure and amino acid sequence in relation to thatstructure in similar polypeptides. In view of that information, oneskilled in the art may predict the alignment of amino acid residues ofhuE3α polypeptide with respect to its three dimensional structure. Oneskilled in the art may choose not to make radical changes to amino acidresidues predicted to be on the surface of the protein, since suchresidues may be involved in important interactions with other molecules.

Additional methods of predicting secondary structure include “threading”(Jones et al., Current Opin. Struct. Biol., 7(3):377–87 (1997); Sippl etal., Structure, 4(1):15–9 (1996)), “profile analysis” (Bowie et al.,Science, 253:164–170 (1991); Gribskov et al., Meth. Enzym., 183:146–159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355–4358(1987)), and “evolutionary linkage” (See Home, supra, and Brenner, supra1997).

Moreover, one skilled in the art may generate test variants containing asingle amino acid substitution at each amino acid residue. The variantscould be screened using activity assays described herein. Such variantscould be used to gather information about suitable variants. Forexample, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

In making such changes, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a its hydropathic index onthe basis of its hydrophobicity and charge characteristics. They are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., J. Mol. Biol., 157: 105–131, 1982). It is knownthat certain amino acids may be substituted for other amino acids havinga similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case.

The U.S. Pat. No. 4,554,101 states that the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein. Asdetailed in U.S. Pat. No. 4,554,101, the following hydrophilicity valueshave been assigned to amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred. One may alsoidentify epitopes from primary amino acid sequences on the basis ofhydrophilicity. Through the methods disclosed in U.S. Pat. No. 4,554,101one of skill in the art is able to identify epitopes from within a givenamino acid sequence. These regions are also referred to as “epitopiccore regions”.

Numerous scientific publications have been devoted to the prediction ofsecondary structure, and to the identification of epitopes, fromanalyses of amino acid sequences. See Chou et al., Biochemistry, 13(2):222–245, 1974; Chou et al., Biochemistry, 113(2): 211–222, 1974; Chou etal., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45–148, 1978; Chou etal., Ann. Rev. Biochem., 47: 251–276 and Chou et al., Biophys. J., 26:367–384, 1979. Moreover, computer programs are currently available toassist with predicting antigenic portions and epitopic core regions ofproteins. Examples include those programs based upon the Jameson-Wolfanalysis (Jameson et al., Comput. Appl. Biosci., 4(1): 181–186, 1998 andWolf et al., Comput. Appl. Biosci., 4(1): 187–191, 1988, the programPepPlot® (Brutlag et al., CABS, 6: 237–245 1990, and Weinberger et al.,Science, 228: 740–742, 1985) and other new programs for protein tertiarystructure prediction (Fetrow et al., Biotechnology, 11: 479–483 1993).

In preferred embodiments, the variants have from 1 to 3, or from 1 to 5,or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, orfrom 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 aminoacid substitutions, insertions, additions and/or deletions, wherein thesubstitutions may be conservative, as described herein, ornon-conservative, or any combination thereof. In addition, the variantscan have additions of amino acid residues either at the carboxy terminusor at the amino terminus (with or without a leader sequence).

Preferred huE3α polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites has been alteredcompared to native huE3α polypeptide. In one embodiment, huE3αpolypeptide variants comprise a greater or a lesser number of N-linkedglycosylation sites. An N-linked glycosylation site is characterized bythe sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X may be any amino acid residue except proline. Thesubstitution(s) of amino acid residues to create this sequence providesa potential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions which eliminate this sequence will removean existing N-linked carbohydrate chain. Also provided is arearrangement of N-linked carbohydrate chains wherein one or moreN-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated. Additional preferred huE3α variants include cysteine variants,wherein one or more cysteine residues are deleted or substituted withanother amino acid (e.g., serine). Cysteine variants are useful whenhuE3α polypeptides must be refolded into a biologically activeconformation such as after the isolation of insoluble inclusion bodies.Cysteine variants generally have fewer cysteine residues than the nativeprotein, and typically have an even number to minimize interactionsresulting from unpaired cysteines.

The term “huE3α fusion polypeptide” refers to a fusion of huE3αI orhuE3αII polypeptide, fragment, and/or variant thereof, with aheterologous peptide or polypeptide. IN addition, the polypeptidecomprising the amino acid sequence of SEQ ID NO: 2 or 4 or huE3αpolypeptide variant many be fused to a homologous polypeptide to form ahomodimer or to a heterologous polypeptide to form a heterodimer.Heterologous peptides and polypeptides include, but are not limited to:an epitope to allow for the detection and/or isolation of a huE3α fusionpolypeptide; a transmembrane receptor protein or a portion thereof, suchas an extracellular domain, or a transmembrane and intracellular domain;a ligand or a portion thereof which binds to a transmembrane receptorprotein; an enzyme or portion thereof which is catalytically active; apolypeptide or peptide which promotes oligomerization, such as a leucinezipper domain; a polypeptide or peptide which increases stability, suchas an immunoglobulin constant region, and a polypeptide which has atherapeutic activity different from the huE3α polypeptide.

In addition, a huE3α polypeptide may be fused to itself or to afragment, variant, or derivative thereof. Fusions can be made either atthe amino terminus or at the carboxy terminus of a huE3α polypeptide.Fusions may be direct with no linker or adapter molecule or indirectusing through a linker or adapter molecule. A linker or adapatermolecule may be one or more amino acid residues, typically from 20 aminoacids residues, or up to about 50 amino acid residues. A linker oradapter molecule may also be designed with a cleavage site for a DNArestriction endonuclease or for a protease to allow for the separationof the fused moieties. It will be appreciated that once constructed, thefusion polypeptides can be derivatized according to the methodsdescribed herein.

In a further embodiment of the invention, a huE3α polypeptide, includinga fragment, variant, and/or derivative, is fused to an Fc region ofhuman IgG. Antibodies comprise two functionally independent parts, avariable domain known as “Fab”, which binds antigens, and a constantdomain known as “Fc”, which is involved in effector functions such ascomplement activation and attack by phagocytic cells. An Fc has a longserum half-life, whereas an Fab is short-lived (Capon et al., Nature,337: 525–31, 1989). When constructed together with a therapeuticprotein, an Fc domain can provide longer half-life or incorporate suchfunctions as Fc receptor binding, protein A binding, complement fixationand perhaps even placental transfer (Capon et al. Nature, 337: 525–31,1989). Table I summarizes the use of certain Fc fusions known in theart, including materials and methods applicable to the production offused huE3α polypeptides.

TABLE I Fc Fusion with Therapeutic Proteins Form of Fusion Fc partnerTherapeutic implications Reference IgG1 N-terminus Hodgkin's disease;U.S. Pat. No. of CD30-L anaplastic lymphoma; T- 5,480,981 cell leukemiaMurine IL-10 anti-inflammatory; Zheng et al., J. Immunol., Fcγ2atransplant rejection 154: 5590–600, 1995 IgG1 TNF receptor septic shockFisher et al., N. Engl. J. Med., 334: 1697–1702, 1996; Van Zee et al., ,J. Immunol., 156: 2221–30, 1996 IgG, IgA, TNF receptor inflammation,U.S. Pat. No. 5,808,029, IgM, or autoimmune disorders issued Sep. 15,1998 IgE (excluding the first domain) IgG1 CD4 receptor AIDS Capon etal., Nature 337: 525–31, 1989 IgG1, N-terminus anti-cancer, antiviralHarvill et al., IgG3 of IL-2 Immunotech., 1: 95–105 1995 IgG1 C-terminusosteoarthritis; WO 97/23614, published of OPG bone density Jul. 3, 1997IgG1 N-terminus anti-obesity PCT/US 97/23183, filed of leptin Dec. 11,1997 Human Ig CTLA-4 autoimmune disorders Linsley, J. Exp. Med., Cγ1174: 561–9, 1991

In one example, all or portion of the human IgG hinge, CH2 and CH3regions may be fused at either the N-terminus or C-terminus of the huE3αpolypeptides using methods known to the skilled artisan. In anotherexample, a portion of a hinge regions and CH2 and CH3 regions may befused. The resulting huE3α Fc-fusion polypeptide may be purified by useof a Protein A affinity column. Peptides and proteins fused to an Fcregion have been found to exhibit a substantially greater half-life invivo than the unfused counterpart. Also, a fusion to an Fc region allowsfor dimerization/multimerization of the fusion polypeptide. The Fcregion may be a naturally occurring Fc region, or may be altered toimprove certain qualities, such as therapeutic qualities, circulationtime, reduce aggregation, etc.

The term “huE3α polypeptide derivatives” refers to huE3αI or huE3αIIpolypeptides, fragments, or variants, as defined herein, that have beenchemically modified. The derivatives are modified in a manner that isdifferent from naturally occurring huE3α, polypeptides either in thetype or location of the molecules attached to the polypeptide.Derivatives may further include molecules formed by the deletion of oneor more chemical groups which are naturally attached to the huE3αpolypeptide.

For example, the polypeptides may be modified by the covalent attachmentof one or more polymers, including, but not limited to, water solublepolymers, N-linked or O-linked carbohydrates, sugars, phosphates, and/orother such molecules. For example, the polymer selected is typicallywater soluble so that the protein to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. The polymer may be of any molecular weight, and may bebranched or unbranched. Included within the scope of suitable polymersis a mixture of polymers. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable.

Suitable water soluble polymers or mixtures thereof include, but are notlimited to, polyethylene glycol (PEG), monomethoxy-polyethylene glycol,dextran (such as low molecular weight dextran, of, for example about 6kD), cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional PEG crosslinking molecules which may be usedto prepare covalently attached huE3α multimers.

For the acylation reactions, the polymer(s) selected should have asingle reactive ester group. For reductive alkylation, the polymer(s)selected should have a single reactive aldehyde group. A reactivealdehyde is, for example, polyethylene glycol propionaldehyde, which iswater stable, or mono C₁–C₁₀ alkoxy or aryloxy derivatives thereof (seeU.S. Pat. No. 5,252,714).

The pegylation of huE3α polypeptides may be carried out by any of thepegylation reactions known in the art, as described for example in thefollowing references: Francis et al., Focus on Growth Factors, 3: 4–10,1992; EP 0154316; EP 0401384 and U.S. Pat. No. 4,179,337. Pegylation maybe carried out via an acylation reaction or an alkylation reaction witha reactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer) as described herein.

Polyethylene glycol (PEG) is a water-soluble polymer suitable for useherein. As used herein, the terms “polyethylene glycol” and “PEG” aremeant to encompass any of the forms of PEG that have been used toderivative proteins, including mono-(C₁–C₁₀)alkoxy- oraryloxy-polyethylene glycol.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated huE3αpolypeptides will generally comprise the steps of (a) reacting thepolypeptide with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby huE3α polypeptidebecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG:protein, thegreater the percentage of poly-pegylated product. In one embodiment, thehuE3α polypeptide derivative may have a single PEG moiety at the aminoterminus. See, for example, U.S. Pat. No. 5,234,784.

Generally, conditions which may be alleviated or modulated by theadministration of the present huE3α polypeptide derivative include thosedescribed herein for huE3α polypeptides. However, the huE3α polypeptidederivative disclosed herein may have additional activities, enhanced orreduced biological activity, or other characteristics, such as increasedor decreased half-life, as compared to the non-derivatized molecules.

The terms “biologically active huE3α polypeptides”, “biologically activehuE3α polypeptide fragments”, “biologically active huE3α polypeptidevariants”, and “biologically active huE3α polypeptide derivatives” referto huE3αI or huE3αII polypeptides having at least one activitycharacteristic of a human E3α ubiquitin ligase, such as the activity ofthe polynucleotide set forth in SEQ ID NOS: 2 or 4. In general, huE3αpolypeptides, fragments, variants, and derivatives thereof, will have atleast one activity characteristic of a huE3α polypeptide such asdepicted in SEQ ID NOS: 2 or 4. In addition, a huE3α polypeptide may beactive as an immunogen, that is, the polypeptide contains at least oneepitope to which antibodies may be raised.

“Naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The term “isolated polypeptide” refers to a polypeptide of the presentinvention that is free from at least one contaminating polypeptide thatis found in its natural environment. Preferably, the isolatedpolypeptide is substantially free from any other contaminating mammalianpolypeptides which would interfere with its therapeutic, preventative,or diagnostic use.

The term “ortholog” refers to a polypeptide that corresponds to apolypeptide identified from a different species that corresponds tohuE3α polypeptide amino acid sequence. For example, mouse and human E3αpolypeptides are considered orthologs.

The term “mature huE3α polypeptide” refers to a polypeptide lacking aleader sequence. A mature polypeptide may also include othermodifications such as proteolytic processing of the amino terminus (withor without a leader sequence) and/or the carboxy terminus, cleavage of asmaller polypeptide from a larger precursor, N-linked and/or O-linkedglycosylation, and the like. An exemplary mature huE3α polypeptide isdepicted by SEQ ID NOS: 2 or 4.

The terms “effective amount” and “therapeutically effective amount”refer to the amount of a huE3α polypeptide or huE3α nucleic acidmolecule used to support an observable level of one or more biologicalactivities of the huE3α polypeptides as set forth herein.

The term “selective binding agent” refers to a molecule or moleculeshaving specificity for huE3α molecules. Selective binding agents includeantibodies, such as polyclonal antibodies, monoclonal antibodies (mAbs),chimeric antibodies, CDR-grafted antibodies, anti-idiotypic (anti-Id)antibodies to antibodies that can be labeled in soluble or bound form,as well as fragments, regions, or derivatives thereof which are providedby known techniques, including, but not limited to enzymatic cleavage,peptide synthesis, or recombinant techniques.

As used herein, the terms, “specific” and “specificity” refer to theability of the selective binding agents to bind to human huE3αpolypeptides. It will be appreciated, however, that the selectivebinding agents may also bind orthologs of huE3α, polypeptides, that is,interspecies versions of E3α, such as mouse and rat E3α polypeptides. Aperferred embodiment relates to antibodies that are highly specific tohuE3α polypeptides yet do not cross-react (that is, they fail to bind)with specificity to non-huE3α polypeptides.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, which is additionally capable of inducing an animal to produceantibodies capable of binding to an epitope of that antigen. An antigencan have one or more epitopes. The specific binding reaction referred toabove is meant to indicate that the antigen will react, in a highlyselective manner, with its corresponding antibody and not with themultitude of other antibodies which can be evoked by other antigens.

Human E3α polypeptides, fragments, variants, and derivatives may be usedto prepare huE3α selective binding agents using methods known in theart. Thus, antibodies and antibody fragments that bind huE3αpolypeptides are within the scope of the present invention. Antibodyfragments include those portions of the antibody which bind to anepitope on the huE3α polypeptide. Examples of such fragments include Faband F(ab′) fragments generated by enzymatic cleavage of full-lengthantibodies. Other binding fragments include those generated byrecombinant DNA techniques, such as the expression of recombinantplasmids containing nucleic acid sequences encoding antibody variableregions. These antibodies may be, for example, polyclonal monospecificpolyclonal, monoclonal, recombinant, chimeric, humanized, human, singlechain, and/or bispecific.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

The term “identity”, as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween nucleic acid molecule or polypeptide sequences, as the case maybe, as determined by the match between strings of two or more nucleotideor two or more amino acid sequences. “Identity” measures the percent ofidentical matches between two or more sequences with gap alignments (ifany) addressed by a particular mathematical model or computer programs(i.e., “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity”, refers to a measure of similarity which includes bothidentical matches and conservative substitution matches. If twopolypeptide sequences have, for example, 10/20 identical amino acids,and the remainder are all non-conservative substitutions, then thepercent identity and similarity would both be 50%. If in the sameexample, there are 5 more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% (15/20). Therefore, in cases where there areconservative substitutions, the degree of similarity between twopolypeptide sequences will be higher than the percent identity betweenthose two sequences.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that (I) has been separated from at leastabout 50 percent of proteins, lipids, carbohydrates or other materialswith which it is naturally found when total DNA is isolated from thesource cells, (2) is not linked to all or a portion of a polynucleotideto which the “isolated nucleic acid molecule” is linked in nature, (3)is operably linked to a polynucleotide which it is not linked to innature, or (4) does not occur in nature as part of a largerpolynucleotide sequence. Preferably, the isolated nucleic acid moleculeof the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, phophylactic orresearch use.

The term “isolated polypeptide” refers to a polypeptide of the presentinvention that (1) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates or other materials with which itis naturally found when isolated from the source cell, (2) is not linked(by covalent or noncovalent interaction) to all or a portion of apolypeptide to which the “isolated polypeptide” is linked in nature, (3)is operably linked (by covalent or noncovalent interaction) to apolypeptide with which it is not linked in nature, or (4) does not occurin nature. Preferably, the isolated polypeptide is substantially freefrom any other contaminating polypeptides or other contaminants that arefound in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic or research use.

The term “conservative amino acid substitution” refers to a substitutionof a native amino acid residue with a normative residue such that thereis little or no effect on the polarity or charge of the amino acidresidue at that position. For example, a conservative substitutionresults from the replacement of a non-polar residue in a polypeptidewith any other non-polar residue. Furthermore, any native residue in thepolypeptide may also be substituted with alanine, as has been previouslydescribed for “alanine scanning mutagenesis.” General rules forconservative amino acid substitutions are set forth in Table II.

TABLE II Amino Acid Substitutions Original Preferred Residues ExemplarySubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe,Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val,Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr,Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala,Norleucine

Conservative amino acid substitutions also encompass non-naturallyoccurring amino acid residues which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics, and other reversed or invertedforms of amino acid moieties. It will be appreciated by those skilled inthe art the nucleic acid and polypeptide molecules described herein maybe chemically synthesized as well as produced by recombinant means.

Conservative modifications to the amino acid sequence (and thecorresponding modifications to the encoding nucleotides) will producehuE3α polypeptides having functional and chemical characteristicssimilar to those of naturally occurring huE3α polypeptides. In contrast,substantial modifications in the functional and/or chemicalcharacteristics of huE3α polypeptides may be accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the molecular backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Naturally occurring residues may be divided intoclasses based on common side chain properties:

-   -   1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;    -   2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   3) acidic: Asp, Glu;    -   4) basic: His, Lys, Arg;    -   5) residues that influence chain orientation: Gly, Pro; and    -   6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human E3α polypeptidethat are homologous with non-human E3α polypeptides, or into thenon-homologous regions of the molecule.

Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods. Such methodsinclude, but are not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J.Applied Math., 48: 1073, 1988.

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,J. Mol. Biol., 215: 403–410, 1990). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., supra). The well known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. A standard comparison matrix (see Dayhoff et al., Atlas ofProtein Sequence and Structure, vol. 5, supp.3 (1978) for the PAM 250comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA, 89:10915–10919, 1992 for the BLOSUM 62 comparison matrix) is also used bythe algorithm.

Preferred parameters for a polypeptide sequence comparison include thefollowing:

-   -   Algorithm: Needleman et al., J. Mol. Biol., 48, 443–453, 1970;    -   Comparison matrix: BLOSUM 62 from Henikoff et al., Proc. Natl.        Acad. Sci. USA, 89: 10915–10919, 1992)    -   Gap Penalty: 12    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisonsinclude the following:

-   -   Algorithm: Needleman et al., J. Mol. Biol., 48: 443–453, 1970;    -   Comparison matrix: matches=+10, mismatch=0    -   Gap Penalty: 50    -   Gap Length Penalty: 3

The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused by those of skill in the art, including those set forth in theProgram Manual, Wisconsin Package, Version 9, September, 1997. Theparticular choices to be made will be apparent to those of skill in theart and will depend on the specific comparison to be made, such as DNAto DNA, protein to protein, protein to DNA; and additionally, whetherthe comparison is between given pairs of sequences (in which case GAP orBestFit are generally preferred) or between one sequence and a largedatabase of sequences (in which case FASTA or BLASTA are preferred).

Synthesis

It will be appreciated by those skilled in the art the nucleic acid andpolypeptide molecules described herein may be produced by recombinantand other means.

Nucleic Acid Molecules

Recombinant DNA methods used herein are generally those set forth inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and/or Ausubelet al., eds., Current Protocols in Molecular Biology, Green PublishersInc. and Wiley and Sons, NY (1994). The present invention provides fornucleic acid molecules as described herein and methods for obtaining themolecules. Human E3α (huE3α) refers to the nucleotide sequence of eitherhuE3αI or huE3αII. A gene or cDNA encoding huE3α polypeptide or fragmentthereof may be obtained by hybridization screening of a genomic or cDNAlibrary, or by PCR amplification. Where a gene encoding a huE3αpolypeptide has been identified from one species, all or a portion ofthat gene may be used as a probe to identify corresponding genes fromother species (orthologs) or related genes from the same species(homologs). The probes or primers may be used to screen cDNA librariesfrom various tissue sources believed to express the huE3α gene. Inaddition, part or all of a nucleic acid molecule having the sequence asset forth in SEQ ID NOS: 1 or 3 may be used to screen a genomic libraryto identify and isolate a gene encoding a huE3α polypeptide. Typically,conditions of moderate or high stringency will be employed for screeningto minimize the number of false positives obtained from the screen.

Nucleic acid molecules encoding huE3α polypeptides may also beidentified by expression cloning which employs the detection of positiveclones based upon a property of the expressed protein. Typically,nucleic acid libraries are screened by the binding of an antibody orother binding partner (e.g., receptor or ligand) to cloned proteinswhich are expressed and displayed on a host cell surface. The antibodyor binding partner is modified with a detectable label to identify thosecells expressing the desired clone.

Additional methods of predicting secondary stricture include “threading”(Jones et al., Current Opin. Struct. Biol., 7(3):377–87 (1997); Sippl etal., Structure, 4(1):15–9 (1996)), “profile analysis” (Bowie et al.,Science, 253:164–170 (1991); Gribskov et al., Meth. Enzym., 183:146–159(1990); Gribskov et al. Proc. Nat. Acad. Sci., 84(13):4355–4358 (1987)),and “evolutionary linkage” (See Home, supra, and Brenner, supra 1997).

Another means of preparing a nucleic acid molecule encoding a huE3αpolypeptide, including a fragment or variant, is chemical synthesisusing methods well known to the skilled artisan such as those describedby Engels et al., Angew. Chem. Intl. Ed., 28: 716–734, 1989. Thesemethods include, inter alia, the phosphotriester, phosphoramidite, andH-phosphonate methods for nucleic acid synthesis. A preferred method forsuch chemical synthesis is polymer-supported synthesis using standardphosphoramidite chemistry. Typically, the DNA encoding the huE3αpolypeptide will be several hundred nucleotides in length. Nucleic acidslarger than about 100 nucleotides can be synthesized as severalfragments using these methods. The fragments can then be ligatedtogether to form the full length huE3α polypeptide. Usually, the DNAfragment encoding the amino terminus of the polypeptide will have anATG, which encodes a methionine residue. This methionine may or may notbe present on the mature form of the huE3α polypeptide, depending onwhether the polypeptide produced in the host cell is designed to besecreted from that cell.

In some cases, it may be desirable to prepare nucleic acid moleculesencoding huE3α polypeptide variants. Nucleic acid molecules encodingvariants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

In certain embodiments, nucleic acid variants contain codons which havebeen altered for the optimal expression of a huE3α polypeptide in agiven host cell. Particular codon alterations will depend upon the huE3αpolypeptide(s) and host cell(s) selected for expression. Such “codonoptimization” can be carried out by a variety of methods, for example,by selecting codons which are preferred for use in highly expressedgenes in a given host cell. Computer algorithms which incorporate codonfrequency tables such as “Ecohigh.cod” for codon preference of highlyexpressed bacterial genes may be used and are provided by the Universityof Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.Other useful codon frequency tables include “Celegans_high.cod”,“Celegans_low.cod”, “Drosophila_high.cod”, “Human_high.cod”,“Maize_high.cod”, and “Yeast_high.cod”.

In other embodiments, nucleic acid molecules encode huE3α variants withconservative amino acid substitutions as described herein, huE3αvariants comprising an addition and/or a deletion of one or moreN-linked or O-linked glycosylation sites, huE3α variants havingdeletions and/or substitutions of one or more cysteine residues, orhuE3α polypeptide fragments as described herein. In addition, nucleicacid molecules may encode any combination of huE3α variants, fragments,and fusion polypeptides described herein.

Vectors and Host Cells

A nucleic acid molecule encoding a huE3α polypeptide is inserted into anappropriate expression vector using standard ligation techniques whereinhuE3α refers to either the polypeptide sequence of huE3αI or huE3αII.The vector is typically selected to be functional in the particular hostcell employed (i.e., the vector is compatible with the host cellmachinery such that amplification of the gene and/or expression of thegene can occur). A nucleic acid molecule encoding a huE3α polypeptidemay be amplified/expressed in prokaryotic, yeast, insect (baculovirussystems), and/or eukaryotic host cells. Selection of the host cell willdepend in part on whether a huE3α polypeptide is to bepost-translationally modified (e.g., glycosylated and/orphosphorylated). If so, yeast, insect, or mammalian host cells arepreferable. For a review of expression vectors, see Meth. Enz., v.185,D. V. Goeddel, ed. Academic Press Inc., San Diego, Calif. (1990).

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotides: a promoter, one or moreenhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide sequence located at the 5′ or 3′ end of the huE3αpolypeptide coding sequence; the oligonucleotide molecule encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus) or myc for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification of the huE3α polypeptide from the host cell. Affinitypurification can be accomplished, for example, by column chromatographyusing antibodies against the tag as an affinity matrix. Optionally, thetag can subsequently be removed from the purified huE3α polypeptide byvarious means such as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), or synthetic, or theflanking sequences may be native sequences which normally function toregulate huE3α polypeptide expression. As such, the source of a flankingsequence may be any prokaryotic or eukaryotic organism, any vertebrateor invertebrate organism, or any plant, provided that the flankingsequences is functional in, and can be activated by, the host cellmachinery.

The flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein other than endogenous huE3α geneflanking sequences will have been previously identified by mappingand/or by restriction endonuclease digestion and can thus be isolatedfrom the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of one ormore flanking sequence may be known. Here, the flanking sequence may besynthesized using the methods described herein for nucleic acidsynthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with suitableoligonucleotide and/or flanking sequence fragments from the same oranother species. Where the flanking sequence is not known, a fragment ofDNA containing a flanking sequence may be isolated from a larger pieceof DNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion to produce the proper DNA fragment followed by isolation usingagarose gel purification, Qiagen® column chromatography (Chatsworth,Calif.), or other methods known to the skilled artisan. The selection ofsuitable enzymes to accomplish this purpose will be readily apparent toone of ordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases, be important for theoptimal expression of the huE3α polypeptide. If the vector of choicedoes not contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (Product No.303-3s, New England Biolabs, Beverly, Mass.) is suitable for mostgram-negative bacteria and various origins (e.g.; SV40, polyoma,adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses such asHPV or BPV) are useful for cloning vectors in mammalian cells.Generally, the origin of replication component is not needed formammalian expression vectors (for example, the SV40 origin is often usedonly because it contains the early promoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene which will beexpressed. Amplification is the process wherein genes which are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whichonly the transformants are uniquely adapted to survive by virtue of theselection gene present in the vector. Selection pressure is imposed byculturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both the selection gene and theDNA that encodes huE3α polypeptides. As a result, increased quantitiesof huE3α polypeptides are synthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the huE3α polypeptide tobe expressed. The Shine-Dalgarno sequence is varied but is typically apolypurine (i.e., having a high A-G content). Many Shine-Dalgarnosequences have been identified, each of which can be readily synthesizedusing methods set forth above and used in a prokaryotic vector.

A leader, or signal, sequence may be used to direct a huE3α polypeptideout of the host cell. Typically, a nucleotide sequence encoding thesignal sequence is positioned in the coding region of the huE3α nucleicacid molecule, or directly at the 5′ end of the huE3α polypeptide codingregion. Many signal sequences have been identified, and any of thosethat are functional in the selected host cell may be used in conjunctionwith the huE3α nucleic acid molecule. Therefore, a signal sequence maybe homologous (naturally occurring) or heterologous to the huE3α gene orcDNA. Additionally, a signal sequence may be chemically synthesizedusing methods described herein. In most cases, the secretion of a huE3αpolypeptide from the host cell via the presence of a signal peptide willresult in the removal of the signal peptide from the huE3α polypeptide.The signal sequence may be a component of the vector, or it may be apart of huE3α DNA that is inserted into the vector.

Included within the scope of this invention is the use of either anucleotide sequence encoding a native huE3α signal sequence joined to ahuE3α polypeptide coding region or a nucleotide sequence encoding aheterologous signal sequence joined to a huE3α polypeptide codingregion. The heterologous signal sequence selected should be one that isrecognized and processed, i.e., cleaved by a signal peptidase, by thehost cell. For prokaryotic host cells that do not recognize and processthe native huE3α signal sequence, the signal sequence is substituted bya prokaryotic signal sequence selected, for example, from the group ofthe alkaline phosphatase, penicillinase, or heat-stable enterotoxin IIleaders. For yeast secretion, the native huE3α signal sequence may besubstituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal sequence issatisfactory, although other mammalian signal sequences may be suitable.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various presequencesto improve glycosylation or yield. For example, one may alter thepeptidase cleavage site of a particular signal peptide, or addpresequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the N-terminus. Alternatively,use of some enzyme cleavage sites may result in a slightly truncatedform of the desired huE3α polypeptide, if the enzyme cuts at such areawithin the mature polypeptide.

In many cases, transcription of a nucleic acid molecule is increased bythe presence of one or more introns in the vector; this is particularlytrue where a polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns used may be naturallyoccurring within the huE3α gene, especially where the gene used is afull length genomic sequence or a fragment thereof. Where the intron isnot naturally occurring within the gene (as for most cDNAs), theintron(s) may be obtained from another source. The position of theintron with respect to flanking sequences and the huE3α gene isgenerally important, as the intron must be expressed to be effective.Thus, when a huE3α cDNA molecule is being expressed, the preferredposition for the intron is 3′ to the transcription start site, and 5′ tothe polyA transcription termination sequence. Preferably, the intron orintrons will be located on one side or the other (i.e., 5′ or 3′) of thecDNA such that it does not interrupt the coding sequence. Any intronfrom any source, including any viral, prokaryotic and eukaryotic (plantor animal) organisms, may be used to practice this invention, providedthat it is compatible with the host cell(s) into which it is inserted.Also included herein are synthetic introns. Optionally, more than oneintron may be used in the vector.

The expression and cloning vectors of the present invention will eachtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding a huE3α polypeptide. Promotersare untranscribed sequences located upstream (5′) to the start codon ofa structural gene (generally within about 100 to 1000 bp) that controlthe transcription and translation of the structural gene. Promoters areconventionally grouped into one of two classes, inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,initiate continual gene product production; that is, there is little orno control over gene expression. A large number of promoters, recognizedby a variety of potential host cells, are well known. A suitablepromoter is operably linked to the DNA encoding a huE3α polypeptide byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.The native huE3α promoter sequence may be used to direct amplificationand/or expression of huE3α DNA. A heterologous promoter is preferred,however, if it permits greater transcription and higher yields of theexpressed protein as compared to the native promoter, and if it iscompatible with the host cell system that has been selected for use.

Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase, atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirsequences have been published, thereby enabling one skilled in the artto ligate them to the desired DNA sequence(s), using linkers or adaptersas needed to supply any useful restriction sites.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowl pox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40). Other suitable mammalian promotersinclude heterologous mammalian promoters, e.g., heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest in controlling huE3α genetranscription include, but are not limited to: the SV40 early promoterregion (Bernoist and Chambon, Nature, 290: 304–310, 1981); the CMVpromoter; the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (Yamamoto et al., Cell, 22: 787–797, 1980); the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA,78: 144–1445, 1981); the regulatory sequences of the metallothioninegene (Brinster et al., Nature, 296: 39–42, 1982); prokaryotic expressionvectors such as the beta-lactamase promoter (Villa-Kamaroff, et al.,Proc. Natl. Acad. Sci. USA, 75: 3727–3731, 1978); or the tac promoter(DeBoer, et al., Proc. Natl. Acad. Sci. USA, 80: 21–25, 1983). Also ofinterest are the following animal transcriptional control regions, whichexhibit tissue specificity and have been utilized in transgenic animals:the elastase I gene control region which is active in pancreatic acinarcells (Swift et al., Cell, 38: 639–646, 1984; Ornitz et al., Cold SpringHarbor Symp. Quant. Biol., 50: 399–409, 1986; MacDonald, Hepatology, 7:425–515, 1987); the insulin gene control region which is active inpancreatic beta cells (Hanahan, Nature, 315: 115–122, 1985); theimmunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., Cell, 38: 647–658 (1984); Adames et al., Nature,318: 533–538 (1985); Alexander et al., Mol. Cell. Biol., 7: 1436–1444,1987); the mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell, 45:485–495, 1986); the albumin gene control region which is active in liver(Pinkert et al., Genes and Devel., 1: 268–276, 1987); thealphafetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol., 5: 1639–1648, 1985; Hammer et al., Science,235: 53–58, 1987); the alpha 1-antitrypsin gene control region which isactive in the liver (Kelsey et al., Genes and Devel., 1: 161–171, 1987);the beta-globin gene control region which is active in myeloid cells(Mogram et al., Nature, 315: 338–340, 1985; Kollias et al., Cell, 46:89–94, 1986); the myelin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., Cell, 48:703–712, 1987); the myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, Nature, 314: 283–286, 1985); and thegonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., Science, 234: 1372–1378, 1986).

An enhancer sequence may be inserted into the vector to increase thetranscription of a DNA encoding a huE3α polypeptide of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10–300 bp in length, that act on the promoter toincrease its transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation of eukaryotic promoters. While an enhancer may be splicedinto the vector at a position 5′ or 3′ to huE3α DNA, it is typicallylocated at a site 5′ from the promoter.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe desired flanking sequences are not already present in the vector,they may be individually obtained and ligated into the vector. Methodsused for obtaining each of the flanking sequences are well known to oneskilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, Carlsbad, Calif.) pBSII (Stratagene Company, La Jolla, Calif.),pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway,N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacII;Invitrogen), pDSR-alpha (PCT Publication No. WO90/14363) andpFastBacDual (Gibco/BRL, Grand Island, N.Y.).

Additional suitable vectors include, but are not limited to, cosmids,plasmids, or modified viruses, but it will be appreciated that thevector system must be compatible with the selected host cell. Suchvectors include, but are not limited to plasmids such as Bluescript®plasmid derivatives (a high copy number ColE1-based phagemid, StratageneCloning Systems Inc., La Jolla Calif.), PCR cloning plasmids designedfor cloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit,PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, Calif.), andmammalian yeast, or virus vectors such is a baculovirus expressionsystem (pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.). Therecombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, or other knowntechniques.

After the vector has been constructed and a nucleic acid moleculeencoding a huE3α polypeptide has been inserted into the proper site ofthe vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. Host cells may beprokaryotic host cells (such as E. coli) or eukaryotic host cells (suchas a yeast cell, an insect cell, or a vertebrate cell). The host cell,when cultured under appropriate conditions, synthesizes a huE3αpolypeptide which can subsequently be collected from the culture medium(if the host cell secretes it into the medium) or directly from the hostcell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity, such as glycosylation or phosphorylation, andease of folding into a biologically active molecule.

A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), 10801Univeristy Boulavard, Manassas, Va. 20110–2209. Examples include, butare not limited to, mammalian cells, such as Chinese hamster ovary cells(CHO) (ATCC No. CCL61) CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad.Sci. USA. 97: 4216–4220, 1980), human embryonic kidney (HEK) 293 or 293Tcells (ATCC No. CRL1573), or 3T3 cells (ATCC No. CCL92). The selectionof suitable mammalian host cells and methods for transformation,culture, amplification, screening and product production andpurification are known in the art. Other suitable mammalian cell lines,are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No.CRL1651), and the CV-1 cell line (ATCC No. CCL70). Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines, which are available from the American Type Culture Collection,Manassas, Va. Each of these cell lines is known by and available tothose skilled in the art of protein expression.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, (ATCC No. 33694) DH5α, DH10, and MC1061 (ATCC No. 53338)) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Psedomonas spp., other Bacillus spp., Streptomyces spp.,and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for the expression of the polypeptides of thepresent invention. Preferred yeast cells include, for example,Saccharomyces cerivisae and Pichia pastoris.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al., Biotechniques, 14: 810–817 (1993); Lucklow, Curr. Opin.Biotechnol., 4: 564–572, 1993; and Lucklow et al. J. Virol., 67:4566–4579, 1993. Preferred insect cells are Sf-9 and Hi5 (Invitrogen,Carlsbad, Calif.).

The transformation of an expression vector for a huE3α polypeptide intoa selected host cell may be accomplished by well known methods includingmethods such as transfection, infection, calcium chloride,electroporation, microinjection, lipofection or the DEAE-dextran methodor other known techinques. The method selected will in part be afunction of the type of host cell to be used. These methods and othersuitable methods are well known to the skilled artisan, and are setforth, for example, in Sambrook et al., supra.

One may also use transgenic animals to express glycosylated huE3αpolypeptides. For example, one may use a transgenic milk-producinganimal (a cow or goat, for example) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to produce huE3αpolypeptides, however, in general, the glycosylation occurring in plantsis different from that produced in mammalian cells, and may result in aglycosylated product which is not suitable for human therapeutic use.

Polypeptide Production

Host cells comprising a huE3α expression vector may be cultured usingstandard media well known to the skilled artisan. The huE3α expressionvector refers a vector which expresses either huE3αI or huE3 αII. Themedia will usually contain all nutrients necessary for the growth andsurvival of the cells. Suitable media for culturing E. coli cellsinclude, for example, Luria Broth (LB) and/or Terrific Broth (TB).Suitable media for culturing eukaryotic cells are, Roswell Park MemorialInstitute medium 1640 (RPMI 1640), Minimal Essential Medium (MEM),and/or Dulbecco's Modified Eagle Medium (DMEM), all of which may besupplemented with serum and/or growth factors as indicated by theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof transformed cells is added as a supplement to the media. The compoundto be used will be dictated by the selectable marker element present onthe plasmid with which the host cell was transformed. For example, wherethe selectable marker element is kanamycin resistance, the compoundadded to the culture medium will be kanamycin. Other compounds forselective growth include ampicillin, tetracycline, and neomycin.

The amount of a huE3α polypeptide produced by a host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, High PerformanceLiquid Chromatography (HPLC) separation, immunoprecipitation, and/oractivity assays such as DNA binding gel shift assays.

If a huE3α polypeptide has been designed to be secreted from the hostcells, the majority of polypeptide maybe found in the cell culturemedium. If however, the huE3α polypeptide is not secreted from the hostcells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for bacterial host cells).

For a huE3α polypeptide situated in the host cell cytoplasm and/ornucleus, the host cells are typically first disrupted mechanically orwith a detergent to release the intracellular contents into a bufferedsolution. Human E3α polypeptide can then be isolated from this solution.

The purification of a huE3α polypeptide from solution can beaccomplished using a variety of techniques. If the polypeptide has beensynthesized such that it contains a tag such as Hexahistidine (huE3αpolypeptide/hexaHis) or other small peptide such as FLAG (Eastman KodakCo., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at eitherits carboxyl or amino terminus, it may essentially be purified in aone-step process by passing the solution through an affinity columnwhere the column matrix has a high affinity for the tag or for thepolypeptide directly (i.e., a monoclonal antibody specificallyrecognizing and binding to the huE3α polypeptide). For example,polyhistidine binds with great affinity and specificity to nickel, thusan affinity column of nickel (such as the Qiagen® nickel columns) can beused for purification of huE3α polypeptide/polyHis. See for example,Ausubel et al., eds., Current Protocols in Molecular Biology, Section10.11.8, John Wiley & Sons, New York (1993).

Where a huE3α polypeptide is prepared without a tag attached, and noantibodies are available, other well known procedures for purificationcan be used. Such procedures include, without limitation, ion exchangechromatography, molecular sieve chromatography, High Performance LiquidChromatography (HPLC), native gel electrophoresis in combination withgel elution, and preparative isoelectric focusing (“Isoprime”machine/technique, Hoefer Scientific, San Francisco, Calif.). In somecases, two or more of these techniques may be combined to achieveincreased purity.

If a huE3α polypeptide is produced intracellularly, the intracellularmaterial (including inclusion bodies for gram-negative bacteria) can beextracted from the host cell using any standard technique known to theskilled artisan. For example, the host cells can be lysed to release thecontents of the periplasm/cytoplasm by French press, homogenization,and/or sonication followed by centrifugation.

If a huE3α polypeptide has formed inclusion bodies in the cytosol, theinclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated at pH extremesor with chaotropic agent such as a detergent, guanidine, guanidinederivatives, urea, or urea derivatives in the presence of a reducingagent such as dithiothreitol at alkaline pH or tris carboxyethylphosphine at acid pH to release, break apart, and solubilize theinclusion bodies. The solubized huE3α polypeptide can then be analyzedusing gel electrophoresis, immunoprecipitation or the like. If it isdesired to isolate the huE3α polypeptide, isolation may be accomplishedusing standard methods such as those described herein and in Marston etal., Meth. Enz., 182: 264–275 1990.

In some cases, a huE3α polypeptide may not be biologically active uponisolation. Various methods for “refolding” or converting the polypeptideto its tertiary structure and generating disulfide linkages, call beused to restore biological activity. Such methods include exposing thesolubilized polypeptide to a pH usually above 7 and in the presence of aparticular concentration of a chaotrope. The selection of chaotrope isvery similar to the choices used for inclusion body solubilization, butusually the chaotrope is used at a lower concentration and is notnecessarily the same as chaotropes used for the solubilization. In mostcases the refolding/oxidation solution will also contain a reducingagent or the reducing agent plus its oxidized form in a specific ratioto generate a particular redox potential allowing for disulfideshuffling to occur in the formation of the protein's cysteine bridge(s).Some of the commonly used redox couples include cysteine/cystamine,glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-2mercaptoethanol(βME)/dithi(βME). A cosolvent may be used to increasethe efficiency of the refolding, and the more common reagents used forthis purpose include glycerol, polyethylene glycol of various molecularweights, arginine and the like.

If inclusion bodies are not formed to a significant degree uponexpression of a huE3α polypeptide, then the polypeptide will be foundprimarily in the supernatant after centrifugation of the cellhomogenate. The polypeptide may be further isolated from the supernatantusing methods such as those described herein.

In situations where it is preferable to partially or completely purify ahuE3α polypeptide such that it is partially or substantially free ofcontaminants, standard methods known to those skilled in the art may beused. Such methods include, without limitation, separation byelectrophoresis followed by electroelution, various types ofchromatography (affinity, immunoaffinity, molecular sieve, and/or ionexchange), and/or high pressure liquid chromatography. In some cases, itmay be preferable to use more than one of these methods for completepurification.

Human E3α polypeptides, including fragments, variants, and/orderivatives thereof may also be prepared by chemical synthesis methods(such as solid phase peptide synthesis) using techniques known in theart, such as those set forth by Merrifield et al., J. Am. Chem. Soc.,85:2149, 1963, Houghten et al., Proc Natl Acad. Sci. USA, 82: 5132 1985,and Stewart and Young, Solid Phase Peptide Synthesis. Pierce ChemicalCo., Rockford, Ill. (1984). Such polypeptides may be synthesized with orwithout a methionine on the amino terminus. Chemically synthesized huE3αpolypeptides may be oxidized using methods set forth in these referencesto form disulfide bridges. Chemically synthesized huE3α polypeptides areexpected to have comparable biological activity to the correspondinghuE3α polypeptides produced recombinantly or purified from naturalsources, and thus may be used interchangeably with a recombinant ornatural huE3α polypeptide.

Another means of obtaining a huE3α polypeptide is via purification frombiological samples such as source tissues and/or fluids in which thehuE3α polypeptide is naturally found. Such purification can be conductedusing methods for protein purification as described herein. The presenceof the huE3α polypeptide during purification may be monitored using, forexample, using an antibody prepared against recombinantly produced huE3αpolypeptide or peptide fragments thereof.

A number of additional methods for producing nucleic acids andpolypeptides are known in the art. See for example, Roberts et al.,Proc. Natl. Acad. Sci U.S.A., 94:12297–12303, 1997, which describes theproduction of fusion proteins between an mRNA and its encoded peptide.See also Roberts, R., Curr. Opin. Chem. Biol., 3:268–273, 1999.Additionally, U.S. Pat. No. 5,824,469 describes methods of obtainingoligonucleotides capable of carrying out a specific biological function.The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those which exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated.

U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483 describeprocesses for producing peptides or polypeptides. This is done byproducing stochastic genes or fragments thereof, and then introducingthese genes into host cells which produce one or more proteins encodedby the stochastic genes. The host cells are then screened to identifythose clones producing peptides or polypeptides having the desiredactivity.

Another method for producing peptides or polypeptides is described inPCT/US98/20094 (WO99/15650) filed by Athersys, Inc. Known as “RandomActivation of Gene Expression for Gene Discovery” (RAGE-GD), the processinvolves the activation of endogenous gene expression or over-expressionof a gene by in situ recombination methods. For example, expression ofan endogenous gene is activated or increased by integrating a regulatorysequence into the target cell which is capable of activating expressionof the gene by non-homologous or illegitimate recombination. The targetDNA is first subjected to radiation, and a genetic promoter inserted.The promoter eventually locates a break at the front of a gene,initiating transcription of the gene. This results in expression of thedesired peptide or polypeptide.

It will be appreciated that these methods can also be used to createcomprehensive IL-17 like protein expression libraries, which cansubsequently be used for high throughput phenotypic screening in avariety of assays, such as biochemical assays, cellular assays, andwhole organism assays (e.g., plant, mouse, etc.).

Chemical Derivatives

Chemically modified derivatives of polypeptides may be prepared by oneskilled in the art, given the disclosures set forth herein below.Polypeptide derivatives are modified in a manner that is different,either in the type or location of the molecules naturally attached tothe polypeptide. Derivatives may include molecules formed by thedeletion of one or more naturally-attached chemical groups. Thepolypeptide comprising the amino acid sequence of SEQ ID NO: 2, or apolypeptide variant, may be modified by the covalent attachment of oneor more polymers. For example, the polymer selected is typically watersoluble so that the protein to which it is attached does not precipitatein an aqueous environment, such as a physiological environment. Includedwithin the scope of suitable polymers is a mixture of polymers.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The polymers each may be of any molecular weight and may be branched orunbranched. The polymers each typically have an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each polymer is preferably between about 5 kDa andabout 50 kDa, more preferably between about 12 kDa and about 40 kDa andmost preferably between about 20 kDa to about 35 kDa. Suitable watersoluble polymers or mixtures thereof include, but are not limited to,N-linked or O-linked carbohydrates; sugars; phosphates; polyethyleneglycol (PEG) (including the forms of PEG that have been used toderivatize proteins, including mono-(C1–C10) alkoxy-oraryloxy-polyethylene glycol), monomethoxy-polyethylene glycol; dextran(such as low molecular weight dextran of, for example about 6 kD,cellulose, or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules which may be used toprepare covalently attached multimers of the polypeptide comprising theamino acid sequence of SEQ ID NO: 2 or a polypeptide variant.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides willgenerally comprise the steps of (a) reacting the polypeptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby thepolypeptide comprising the amino acid sequence of SEQ ID NO: 2, or apolypeptide variant becomes attached to one or more polymer molecules,and (b) obtaining the reaction product(s). The optimal reactionconditions will be determined based on known parameters and the desiredresult. For example, the larger the ratio of polymer molecules:protein,the greater the percentage of attached polymer molecule. In oneembodiment, the polypeptide derivative may have a single polymermolecule moiety at the amino terminus. (See, for example, U.S. Pat. No.5,234,784). The pegylation of the polypeptide may be specificallycarried out by any of the pegylation reactions known in the art, asdescribed for example in the following references: Francis et al., Focuson Growth Factors, 3:4–10 (1992); EP 0154316; EP 0401384 and U.S. Pat.No. 4,179,337. For example, pegylation may be carried out via anacylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer) as described herein. For the acylation reactions, thepolymer(s) selected should have a single reactive ester group. Forreductive alkylation, the polymer(s) selected should have a singlereactive aldehyde group. A reactive aldehyde is, for example,polyethylene glycol propionaldehyde, which is water stable, or monoC1–C10 alkoxy or aryloxy derivatives thereof (see U.S. Pat. No.5,252,714).

In another embodiment, polypeptides may be chemically coupled to biotin,and the biotin/polypeptide molecules which are conjugated are thenallowed to bind to avidin, resulting in tetravalentavidin/biotin/polypeptide molecules. Polypeptides may also be covalentlycoupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resultingconjugates precipitated with anti-DNP or anti-TNP-IgM to form decamericconjugates with a valency of 10.

Generally, conditions which may be alleviated or modulated by theadministration of the present polypeptide derivatives include thosedescribed herein for polypeptides. However, the polypeptide derivativesdisclosed herein may have additional activities, enhanced or reducedbiological activity, or other characteristics, such as increased ordecreased half-life, as compared to the non-derivatized molecules.

Selective Binding Agents

As used herein, ther term “selective binding agent” refers to a moleculewhich has specificity for one or more huE3α polypeptides. Suitableselective binding agents include, but are not limited to, antibodies andderivatives thereof, polypeptides, and small molecules. Suitableselective binding agents may be prepared using methods known in the art.An exemplary huE3α polypeptide selective binding agent of the presentinvention is capable of binding a certain portion of the huE3αpolypeptide thereby inhibiting the binding of a cofactor to the huE3αpolypeptide.

Human E3α polypeptides, fragments, variants, and derivatives may be usedto prepare selective binding agents (such as antibodies) using methodsknown in the art; wherein huE3α polypeptide refers to either huE3αI orhuE3αII polypeptide. Thus, selective binding agents such as antibodiesand antibody fragments that bind huE3α polypeptides are within the scopeof the present invention. The antibodies may be polyclonal, monospecificpolyclonal, monoclonal, recombinant, chimeric, humanized human, singlechain, and/or bispecific.

Polyclonal antibodies directed toward a huE3α polypeptide generally areraised in animals (e.g., rabbits or mice) by multiple subcutaneous orintraperitoneal injections of huE3α polypeptide and an adjuvant. Itmaybe useful to conjugate a huE3α polypeptide, or a variant, fragment,or derivative thereof to a carrier protein that is immunogenic in thespecies to be immunized, such as keyhole limpet heocyanin, serum,albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also,aggregating agents such as alum are used to enhance the immune response.After immunization, the animals are bled and the serum is assayed foranti-huE3α antibody titer.

Monoclonal antibodies directed toward huE3α polypeptides are producedusing any method which provides for the production of antibody moleculesby continuous cell lines in culture. Examples of suitable methods forpreparing monoclonal antibodies include the hybridoma methods of Kohleret al., Nature, 256: 495–497, 1975 and the human B-cell hybridomamethod, Kozbor, J. Immunol., 133: 3001, 1984; Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51–63 (MarcelDekker, Inc., New York, 1987). Also provided by the invention arehybridoma cell lines which produce monoclonal antibodies reactive withhuE3α polypeptides.

Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy and/or light chain is identical with or homologous to acorresponding sequence in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity. See, U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci., 81: 6851–6855 (1985).

In another embodiment, a monoclonal antibody of the invention is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.Humanization can be performed following methods known in the art (Joneset al., Nature 321: 522–525, 1986; Riechmann et al., Nature, 332:323–327 (1988); Verhoeyen et al., Science 239:1534–1536 1988), bysubstituting rodent complementarity-determining regions (CDRs) for thecorresponding regions of a human antibody.

Also encompassed by the invention are human antibodies which bind huE3αpolypeptides, fragments, variants and/or derivatives. Such antibodiesare produced by immunization with a huE3α antigen (i.e., having at least6 contiguous amino acids), optionally conjugated to a carrier, oftransgenic animals (e.g., mice) that are capable of producing arepertoire of human antibodies in the absence of endogenousimmunoglobulin production. See, for example, Jakobovits et al., Proc.Natl. Acad. Sci., 90: 2551–2555, 1993; Jakobovits et al., Nature 362:255–258, 1993; Bruggermann et al., Year in Immuno., 7: 33 (1993). In onemethod, such transgenic animals are produced by incapacitating theendogenous loci encoding the heavy and light immunglobulin chainstherein, and inserting loci encoding human heavy and light chainproteins into the genome thereof. Partially modified animals, that isthose having less than the full complement of modifications, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies with human variable regions, including human (ratherthan e.g., murine) antibodies which are immunospecific for theseantigens. See PCT application Nos. PCT/US96/05928 and PCT/US93/06926.Additional methods are described in U.S. Pat. No. 5,545,807, PCTapplication Nos. PCT/US91/245, PCT/GB89/01207, and in EP 546073B1 and EP546073A1.

Human antibodies can also be produced from phage-display libraries(Hoogenboom et al., J. Mol. Biol. 227: 381 1991; Marks et al., J. Mol.Biol. 222: 581, 1991). These processes mimic immune selection throughthe display of antibody repertoires on the surface of filamentousbacteriophage, and subsequent selection of phage by their binding to anantigen of choice. One such technique is described in PCT ApplicationWO99/10494, filed in the name of Adams et al., which describes theisolation of high affinity and functional agonistic antibodies for MPL-and msk-receptors using such an approach.

Chimeric, CDR grafted, and humanized antibodies are typically producedby recombinant methods. Nucleic acids encoding the antibodies areintroduced into host cells and expressed using materials and proceduresdescribed herein. In a preferred embodiment, the antibodies are producedin mammalian host cells, such as CHO cells. Human antibodies may beproduced by the expression of recombinant DNA in host cells or byexpression in hybridoma cells as described herein.

For diagnostic applications, in certain embodiments, anti-huE3αantibodies typically will be labeled with a detectable moiety. Thedetectable moiety can be any one which is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the detectablemoiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, afluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, β-galactosidase, or horseradish peroxidase (Bayer et al.,Meth. Enz., 184: 138–163 1990).

The anti-huE3α antibodies of the invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Sola, MonoclonalAnitibodies: A Manual of Techniques, pp. 147–158 (CRC Press, Inc.,1987)) for the detection and quantitation of huE3α polypeptides. Theantibodies will bind huE3α polypeptides with an affinity which isappropriate for the assay method being employed.

Competitive binding assays rely on the ability of a labeled standard(e.g., a huE3α polypeptide, or an immunologically reactive portionthereof) to compete with the test sample analyte (a huE3α polypeptide)for binding with a limited amount of antihuE3α antibody. The amount of ahuE3α polypeptide in the test sample is inversely proportional to theamount of standard that becomes bound to the antibodies. To facilitatedetermining the amount of standard that becomes bound, the antibodiestypically are insolubilized before or after the competition, so that thestandard and analyte that are bound to the antibodies may convenientlybe separated from the standard and analyte which remain unbound.

Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte is typically bound by a first antibody which isimmobilized on a solid support, and thereafter a second antibody bindsto the analyte, thus forming an insoluble three part complex. See, e.g.,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme-linked immunosorbent assay (ELISA), in which case thedetectable moiety is an enzyme.

The selective binding agents, including antibodies, are also useful forin vivo imaging. An antibody labeled with a detectable moiety may beadministered to an animal, preferably into the bloodstream, and thepresence and location of the labeled antibody in the host is assayed.The antibody may be labeled with any moiety that is detectable in ananimal, whether by nuclear magnetic resonance, radiology, or otherdetection means known in the art.

Selective binding agents of the invention, including antibodies, may beused as therapeutics. These therapeutic agents are generally agonists orantagonists, in that they either enhance or reduce, respectively, atleast one of the biological activities of a polypeptide. In oneembodiment, antagonist antibodies of the invention are antibodies orbinding fragments thereof which are capable of specifically binding to apolypeptide and which are capable of inhibiting or eliminating thefunctional activity of a polypeptide in vivo or in vitro. In preferredembodiments, the selective binding agent, e.g., an antagonist antibodywill inhibit the functional activity of a polypeptide by at least about50%, and preferably by at least about 80%. In another embodiment, theselective binging agent may be an antibody that is capable ofinteracting with a binding partner (a ligand, co-factor, or receptor)thereby inhibiting or eliminating activity in vitro or in vivo.Selective binding agents, including agonist and antagonist antibodiesare identified by screening assays which are well known in the art.

The invention also relates to a kit comprising huE3α selective bindingagents (such as antibodies) and other reagents useful for detectinghuE3α levels in biological samples. Such reagents may include asecondary activity, a detectable label, blocking serum, positive andnegative control samples, and detection reagents.

Genetically Engineered Non-Human Animals

Additionally included within the scope of the present invention arenon-human animals such as mice, rats, or other rodents, rabbits, goats,or sheep, or other farm animals, in which the gene (or genes) encoding anative E3α ubiquitin ligase polypeptide (such as E3αI or E3αII) has(have) been disrupted (“knocked out”) such that the level of expressionof this gene or genes is (are) significantly decreased or completelyabolished. Such animals may be prepared using techniques and methodssuch as those described in U.S. Pat. No. 5,557,032.

The present invention further includes non-human animals such as mice,rats, or other rodents, rabbits, goats, or sheep, or other farm animals,in which either the native form of the E3α ubiquitin ligase polypeptidegene(s) for that animal or a heterologous E3α ubiquitin ligasepolypeptide gene(s) is (are) over expressed by the animal, therebycreating a “transgenic” animal. Such transgenic animals may be preparedusing well known methods such as those described in U.S. Pat. No.5,489,743 and PCT application No. WO94/28122.

The present invention further includes non-human animals in which thepromoter for one or more of the E3α ubiquitin ligase polypeptides of thepresent invention is either activated or inactivated (e.g., by usinghomologous recombination methods) to alter the level of expression ofone or more of the native E3α ubiquitin ligase polypeptides.

These non-human animals may be used for drug candidate screening. Insuch screening, the impact of a drug candidate on the animal may bemeasured. For example, drug candidates may decrease or increase theexpression of the E3α ubiquitin ligase polypeptide gene. In certainembodiments, the amount of E3α ubiquitin ligase polypeptide, or afragment(s), that is produced may be measured after the exposure of theanimal to the drug candidate. Additionally, in certain embodiments, onemay detect the actual impact of the drug candidate on the animal. Forexample, the overexpression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct or its ability to prevent or inhibit a pathological condition.

Microarray

It will be appreciated that DNA microarray technology can be utilized inaccordance with the present invention. DNA microarrays are miniature,high density arrays of nucleic acids positioned on a solid support, suchas glass. Each cell or element within the array has numerous copies of asingle species of DNA which acts as a target for hybridization for itscognate mRNA. In expression profiling using DNA microarray technology,mRNA is first extracted from a cell or tissue sample and then convertedenzymatically to fluorescently labeled cDNA. This material is hybridizedto the microarray and unbound cDNA is removed by washing. The expressionof discrete genes represented on the array is then visualized byquantitating the amount of labeled cDNA which is specifically bound toeach target DNA. In this way, the expression of thousands of genes canbe quantitated in a high throughput, parallel manner from a singlesample of biological material.

This high throughput expression profiling has a broad range ofapplications with respect to the molecules of the invention, including,but not limited to: the identification and validation of disease-relatedgenes as targets for therapeutics; molecular toxicology of molecules andinhibitors thereof; stratification of populations and generation ofsurrogate markers for clinical trials; and the enhancement of a relatedsmall molecule drug discovery by aiding in the identification ofselective compounds in high throughput screens (HTS).

Assaying for Other Modulators of huE3α Polypeptide Activity

In some situations, it may be desirable to identify molecules that aremodulators, i.e., antagonists and agonists, of the activity of huE3αpolypeptide.

Natural or synthetic molecules that modulate huE3α polypeptides can beidentified using one or more screening assays, such as those describedherein. Such molecules may be administered either in an ex vivo manner,or in an in vivo manner by injection, or by oral delivery, implantationdevice, or the like.

The following definition is used herein for describing the assays. “Testmolecule(s)” refers to the molecule(s) that is/are under evaluation forthe ability to modulate (i.e., increase or decrease) the activity of ahuE3α polypeptide. Most commonly, a test molecule will interact directlywith a huE3α polypeptide. However, it is also contemplated that a testmolecule may also modulate huE3α polypeptide activity indirectly, suchas by affecting huE3α gene expression, or by binding to a huE3α bindingpartner (e.g., receptor, co-factor or ligand). In one embodiment, a testmolecule will bind to a huE3α polypeptide with an affinity constant ofat least about 10⁻⁶ M, preferably about 10⁻⁸ M, more preferably about10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

Methods for identifying compounds which interact with huE3α polypeptidesare encompassed by the present invention. In certain embodiments, ahuE3α polypeptide is incubated with a test molecule under conditionswhich permit the interaction of the test molecule with a huE3αpolypeptide, and the extent of the interaction can be measured. The testmolecule(s) can be screened in a substantially purified form or in acrude mixture. Test molecule(s) can be nucleic acid molecules, proteins,peptides, carbohydrates, lipids, or small molecular weight organic orinorganic compounds. Once a set of has been identified as interactingwith a huE3α polypeptide, the molecules may be further evaluated fortheir ability to increase or decrease huE3α activity.

The measurement of the interaction of test molecules with huE3αpolypeptides may be carried out in several formats, including cell-basedbinding assays, membrane binding assays, solution-phase assays andimmunoassays. In general, test molecules are incubated with a huE3αpolypeptide for a specified period of time, and huE3α activity isdetermined by one or more assays described herein for measuringbiological activity.

The interaction of test molecules with huE3α polypeptides may also beassayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of huE3α polypeptidescontaining epitope tags as described herein may be used in solution andimmunoassays.

In certain embodiments, a huE3α polypeptide agonist or antagonist may bea protein, peptide, carbohydrate, lipid, or small molecular weightmolecule which interacts with huE3α polypeptide to regulate itsactivity. Molecules which regulate huE3α polypeptide expression includenucleic acids which are complementary to nucleic acids encoding a huE3αpolypeptide, or are complementary to nucleic acids sequences whichdirect or control the expression of huE3α polypeptide, and which act asantisense regulators of expression.

Once a set of test molecules has been identified as interacting with apolypeptide, the molecules may be further evaluated for their ability toincrease or decrease polypeptide activity. The measurement of theinteraction of test molecules with polypeptides may be carried out inseveral formats, including cell-based binding assays, membrane bindingassays, solution-phase assays and immunoassays. In general, testmolecules are incubated with a polypeptide for a specified period oftime, and polypeptide activity is determined by one or more assays formeasuring biological activity.

The interaction of test molecules with polypeptides may also be assayeddirectly using polyclonal or monoclonal antibodies in an immunoassay.Alternatively, modified forms of polypeptides containing epitope tags asdescribed herein may be used in immunoassays.

In the event that polypeptides display biological activity through aninteraction with a binding partner (e.g., a receptor, a ligand or aco-factor), a variety of in vitro assays may be used to measure thebinding of a polypeptide to the corresponding binding partner (such as aselective binding agent, receptor, ligand, or co-factor). These assaysmay be used to screen test molecules for their ability to increase ordecrease the rate and/or the extent of binding of a polypeptide to itsbinding partner. In one assay, a polypeptide is immobilized in the wellsof a microtiter plate. Radiolabeled binding partner (for example,iodinated binding partner) and the test molecule(s) can then be addedeither one at a time (in either order) or simultaneously to the wells.After incubation, the wells can be washed and counted using ascintillation counter, for radioactivity to determine the extent towhich the binding partner bound to polypeptide. Typically, the moleculeswill be tested over a range of concentrations, and a series of controlwells lacking one or more elements of the test assays can be used foraccuracy in the evaluation of the results. An alternative to this methodinvolves reversing the “positions” of the proteins, i.e., immobilizingbinding partner to the microtiter plate wells, incubating with the testmolecule and radiolabeled polypeptide, and determining the extent ofpolypeptide binding. See, for example, Chapter 18, Current Protocols inMolecular Biology, Ausubel et at., eds., John Wiley & Sons, New York,N.Y. (1995).

As an alternative to radiolabelling, a polypeptide or its bindingpartner may be conjugated to biotin and the presence of biotinylatedprotein can then be detected using streptavidin linked to an enzyme,such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), thatcan be detected colorometrically, or by fluorescent tagging ofstreptavidin. An antibody directed to a polypeptide or to a bindingpartner and conjugated to biotin may also be used and can be detectedafter incubation with enzyme-linked streptavidin linked to AP or HRP.

A polypeptide or a like binding partner can also be immobilized byattachment to agarose beads, acrylic beads or other types of such inertsolid phase substrates. The substrate-protein complex can be placed in asolution containing the complementary protein and the test compound.After incubation, the beads can be precipitated by centrifugation, andthe amount of binding between a polypeptide and its binding partner canbe assessed using the methods described herein. Alternatively, thesubstrate-protein complex can be immobilized in a column, and the testmolecule and complementary protein are passed through the column. Theformation of a complex between a polypeptide and its binding partner canthen be assessed using any of the techniques set forth herein, i.e.,radiolabelling, antibody binding or the like.

Another in vitro assay that is useful for identifying a test moleculewhich increases or decreases the formation of a complex between apolypeptide and a binding partner is a surface plasmon resonancedetector system such as the BIAcore assay system (Pharmacia, Piscataway,N.J.). The BIAcore system may be carried out using the manufacturer'sprotocol. This assay essentially involves the covalent binding of eitherpolypeptide or a binding partner to a dextran-coated sensor chip whichis located in a detector. The test compound and the other complementaryprotein can then be injected, either simultaneously or sequentially,into the chamber containing the sensor chip. The amount of complementaryprotein that binds can be assessed based on the change in molecular masswhich is physically associated with the dextran-coated side of thesensor chip; the change in molecular mass can be measured by thedetector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease theformation of a complex between a polypeptide and a binding partner. Inthese cases, the assays set forth herein can be readily modified byadding such additional test compound(s) either simultaneous with, orsubsequent to, the first test compound. The remainder of the steps inthe assay are set forth herein.

In vitro assays such as those described herein may be usedadvantageously to screen large numbers of compounds for effects oncomplex formation by polypeptide and binding partner. The assays may beautomated to screen compounds generated in phage display, syntheticpeptide, and chemical synthesis libraries.

Compounds which increase or decrease the formation of a complex betweena polypeptide and a binding partner may also be screened in cell cultureusing cells and cell lines expressing either polypeptide or bindingpartner. Cells and cell lines may be obtained from any mammal, butpreferably will be from human or other primate, canine, or rodentsources. The binding of a polypeptide to cells expressing bindingpartner at the surface is evaluated in the presence or absence of testmolecules, and the extent of binding may be determined by, for example,flow cytometry using a biotinylated antibody to a binding partner. Cellculture assays can be used advantageously to further evaluate compoundsthat score positive in protein binding assays described herein.

Cell cultures can be used to screen the impact of a drug candidate. Forexample, drug candidates may decrease or increase the expression of thehuE3α polypeptide gene. In certain embodiments, the amount of huE3αpolypeptide or a fragment(s) that is produced may be measured afterexposure of the cell culture to the drug candidate. In certainembodiments, one may detect the actual impact of the drug candidate onthe cell culture. For example, the overexpression of a particular genemay have a particular impact on the cell culture. In such cases, one maytest a drug candidate's ability to increase or decrease the expressionof the gene or its ability to prevent or inhibit a particular impact onthe cell culture. In other examples, the production of a particularmetabolic product such as a fragment of a polypeptide, may result in, orbe associated with, a disease or pathological condition. In such cases,one may test a drug candidate's ability to decrease the production ofsuch a metabolic product in a cell culture.

A yeast two hybrid system (Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578–9583, 1991) can be used to identify novel polypeptides that bind toa yeast-two hybrid bait construct can be generated in a vector (such asthe pAS2-1 form Clontech) which encodes a yeast-two hybrid domain fusedto the huE3α polynucleotide. This bait construct may be used to screenhuman cDNA libraries wherein the cDNA library sequences are fused toGAL4 activation domains. Positive interactions will result in theactivation of a reporter gene such as β-gal. Positive clones emergingfrom the screening may be characterized further to identify interactingproteins.

Internalizing Proteins

The TAT protein sequence (from HIV) can be used to internalize proteinsinto a cell by targeting the lipid bi-layer component of the cellmembrane. See e.g., Falwell et al., Proc. Natl. Acad. Sci., 91: 664–668,1994. For example, an 11 amino acid sequence (YGRKKRRQRRR; SEQ ID NO:16) of the HIV TAT protein (termed the “protein transduction domain”, orTAT PDT) has been shown to mediate delivery of large bioactive proteinssuch as β-galactosidase and p27Kip across the cytoplasmic membrane andthe nuclear membrane of a cell. See Schwarze et al., Science, 285:1569–1572, 1999; and Nagahara et al., Nature Medicine, 4: 1449–1452,1998. Schwartze et al. (Science, 285: 1569–72, 1999) demonstrated thatcultured cells acquired β-gal activity when exposed to a fusion of theTAT PDT and β-galactosidase. Injection of mice with the TAT-β-gal fusionproteins resulted in β-gal expression in a number of tissues, includingliver, kidney, lung, heart, and brain tissue.

It will thus be appreciated that the TAT protein sequence may be used tointernalize a desired protein or polypeptide into a cell. In the contextof the present invention, the TAT protein sequence can be fused toanother molecule such as a huE3α antagonist (i.e.: anti-huE3α selectivebinding agent or small molecule) and administered intracellularly toinhibit the activity of the huE3α molecule. Where desired, the huE3αprotein itself, or a peptide fragment or modified form of huE3α, may befused to such a protein transducer for administrating to cells using theprocedures, described above.

Therapeutic Uses

The huE3α nucleic acid molecules, polypeptides, and antagonists thereof(including, but not limited to, anti-huE3α selective binding agents) canbe used to treat, diagnose, and/or prevent a number of diseases,conditions, and disorders, including but not limited to cachexia, musclewasting diseases and other catabolic disorders such as cancer cachexia,renal cachexia, inflammatory cachexia, muscle wasting disordersassociated with metabolic acidosis, uremia, burns, hyperthyroidism,Cushing's syndrome and fasting, and denervation atrophy, diabetesmellitus, sepsis and AIDS wasting syndrome.

Those skilled in the art will recognize that many combinations ofdeletions, insertions, and substitutions (individually or collectively“variant(s)” herein) can be made within the amino acid sequences of thehuE3α polypeptide, provided that the resulting molecule is biologicallyactive (e.g., possesses the ability to affect one or more of thediseases and disorders such as those recited herein).

As contemplated by the present invention, a polypeptide, or antagonistthereof (including, but not limited to, anti-huE3α selective bindingagents) may be administered as an adjunct to other therapy and also withother pharmaceutical compositions suitable for the indication beingtreated. A polypeptide and any of one or more additional therapies orpharmaceutical formulations may be administered separately,sequentially, or simultaneously.

In a specific embodiment, the present invention is directed to the useof a huE3α polypeptide, or antagonist (including, but not limited to,anti-huE3α selective binding agents) thereof in combination(pretreatment, post-treatment, or concurrent treatment) with secreted orsoluble human fas antigen or recombinant versions thereof (WO96/20206and Mountz et al., J. Immunology, 155: 4829–4837; and EP 510 691.WO96/20206 discloses secreted human fas antigen (native and recombinant,including an Ig fusion protein), methods for isolating the genesresponsible for coding the soluble recombinant human fas antigen,methods for cloning the gene in suitable vectors and cell types, andmethods for expressing the gene to produce the inhibitors. EP 510 691teaches DNAs coding for human fas antigen including soluble fas antigen,vectors expressing for said DNAs and transformants transfected with thevector. When administered parenterally, doses of a secreted or solublefas antigen fusion protein each are generally from about 1 microgram/kgto about 100 micrograms/kg.

Treatment of the diseases and disorders recited herein can include theuse of first line drugs for control of pain and inflammation; thesedrugs are classified as non-steroidal, anti-inflammatory drugs (NSAIDs).Secondary treatments include corticosteroids, slow acting antirheumaticdrugs (SAARDs), or disease modifying (DM) drugs. Information regardingthe following compounds can be found in The Merck Manual of Diagnosisand Therapy, Sixteenth Edition, Merck, Sharp & Dohme ResearchLaboratories, Merck & Co., Rahway, N.J. (1992) and in Pharmaprojects,PJB Publications Ltd.

In a specific embodiment, the present invention is directed to the useof a huE3α, or antagonist (including, but not limited to, anti-huE3αselective binding agents) and any of one or more NSAIDs for thetreatment of the diseases and disorders recited herein. NSAIDs owe theiranti-inflammatory action, at least in part, to the inhibition ofprostaglandin synthesis (Goodman and Gilman in “The PharmacologicalBasis of Therapeutics,” MacMillan 7th Edition (1985)). NSAIDs can becharacterized into at least nine groups: (1) salicylic acid derivatives;(2) propionic acid derivatives; (3) acetic acid derivatives; (4) fenamicacid derivatives; (5) carboxylic acid derivatives; (6) butyric acidderivatives; (7) oxicams; (8) pyrazoles and (9) pyrazolones.

In another specific embodiment, the present invention is directed to theuse of an huE3α polypeptide, or antagonist (including, but not limitedto, anti-huE3α selective binding agents) in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moresalicylic acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. Such salicylic acid derivatives, prodrugesters and pharmaceutically acceptable salts thereof comprise:acetaminosalol, aloxiprin, aspirin, benorylate, bromosaligenin, calciumacetylsalicylate, choline magnesium trisalicylate, magnesium salicylate,choline salicylate, diflusinal, etersalate, fendosal, gentisic acid,glycol salicylate, imidazole salicylate, lysine acetylsalicylate,mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine,parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide,salicylamide O-acetic acid, salsalate, sodium salicylate andsulfasalazine. Structurally related salicylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In an additional specific embodiment, the present invention is directedto the use of an huE3α polypeptide, or antagonist (including, but notlimited to, anti-huE3α selective binding agents) in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more propionic acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. The propionic acid derivatives, prodrugesters, and pharmaceutically acceptable salts thereof comprise:alminoprofen, benoxaprofen, bucloxic acid, carprofen, dexindoprofen,fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, furcloprofen,ibuprofen, ibuprofen aluminum, ibuproxam, indoprofen, isoprofen,ketoprofen, loxoprofen, miroprofen, naproxen, naproxen sodium,oxaprozin, piketoprofen, pimeprofen, pirprofen, pranoprofen, protizinicacid, pyridoxiprofen, suprofen, tiaprofenic acid and tioxaprofen.Structurally related propionic acid derivatives having similar analgesicand anti-inflammatory properties are also intended to be encompassed bythis group.

In yet another specific embodiment, the present invention is directed tothe use of a huE3α polypeptide, or antagonist (including, but notlimited to, anti-huE3α selective binding agents) in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more acetic acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. The acetic acid derivatives, prodrug esters,and pharmaceutically acceptable salts thereof comprise: acemetacin,alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin,diclofenac potassium, diclofenac sodium, etodolac, felbinac,fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac,glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac,metiazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin,sulindac, talmetacin, tiaramide, tiopinac, tolmetin, tolmetin sodium,zidometacin and zomepirac. Structurally related acetic acid derivativeshaving similar analgesic and anti-inflammatory properties are alsointended to be encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of a huE3α polypeptide, or antagonist (including, but not limitedto, anti-huE3α selective binding agents) in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more fenamicacid derivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The fenamic acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof comprise: enfenamic acid,etofenamate, flufenamic acid, isonixin, meclofenamic acid, meclofenamatesodium, medofenamic acid, mefenamic acid, niflumic acid, talniflumate,terofenamate, tolfenamic acid and ufenamate. Structurally relatedfenamic acid derivatives having similar analgesic and anti-inflammatoryproperties are also intended to be encompassed by this group.

In an additional specific embodiment, the present invention is directedto the use of a huE3α polypeptide, or antagonist (including, but notlimited to, anti-huE3α selective binding agents) in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more carboxylic acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. The carboxylic acid derivatives, prodrugesters, and pharmaceutically acceptable salts thereof which can be usedcomprise: clidanac, difluisal, flufenisal, inoridine, ketorolac andtinoridine. Structurally related carboxylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In yet another specific embodiment, the present invention is directed tothe use of a huE3α polypeptide, or antagonist (including, but notlimited to, anti-huE3α selective binding agents) in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more butyric acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. The butyric acid derivatives, prodrug esters,and pharmaceutically acceptable salts thereof comprise: bumadizon,butibufen, fenbufen and xenbucin. Structurally related butyric acidderivatives having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of a huE3α polypeptide, or antagonist (including, but not limitedto, anti-huE3α selective binding agents) in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moreoxicams, prodrug esters, or pharmaceutically acceptable salts thereof.The oxicams, prodrug esters, and pharmaceutically acceptable saltsthereof comprise: droxicam, enolicam, isoxicam, piroxicam, sudoxicam,tenoxicam and 4-hydroxyl-1,2-benzothiazine 1,1-dioxide4-(N-phenyl)-carboxamide. Structurally related oxicams having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

In still another specific embodiment, the present invention is directedto the use of a huE3α polypeptide, or antagonist (including, but notlimited to, anti-huE3α selective binding agents) in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more pyrazoles, prodrug esters, or pharmaceutically acceptable saltsthereof. The pyrazoles, prodrug esters, and pharmaceutically acceptablesalts thereof which may be used comprise: difenamizole and epirizole.Structurally related pyrazoles having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In an additional specific embodiment, the present invention is directedto the use of a huE3α polypeptide, or antagonist (including, but notlimited to, anti-huE3α selective binding agents) in combination(pretreatment, post-treatment or, concurrent treatment) with any of oneor more pyrazolones, prodrug esters, or pharmaceutically acceptablesalts thereof. The pyrazolones, prodrug esters and pharmaceuticallyacceptable salts thereof which may be used comprise: apazone,azapropazone, benzpiperylon, feprazone, mofebutazone, morazone,oxyphenbutazone, phenylbutazone, pipebuzone, propylphenazone,ramifenazone, suxibuzone and thiazolinobutazone. Structurally relatedpyrazalones having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of a huE3α polypeptide, or antagonist (including, but not limitedto, anti-huE3α selective binding agents) in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more of thefollowing NSAIDs: e-acetamidocaproic acid, S-adenosyl-methionine,3-amino-4-hydroxybutyric acid, amixetrine, anitrazafen, antrafenine,bendazac, bendazac lysinate, benzydamine, beprozin, broperamole,bucolome, bufezolac, ciproquazone, cloximate, dazidamine, deboxamet,detomidine, difenpiramide, difenpyramide, difisalamine, ditazol,emorfazone, fanetizole mesylate, fenflumizole, floctafenine, flumizole,flunixin, fluproquazone, fopirtoline, fosfosal, guaimesal, guaiazolene,isonixirn, lefetamine HCl, leflunomide, lofemizole, lotifazole, lysinclonixinate, meseclazone, nabumetone, nictindole, nimesulide, orgotein,orpanoxin, oxaceprol, oxapadol, paranyline, perisoxal, perisoxalcitrate, pifoxime, piproxen, pirazolac, pirfenidone, proquazone,proxazole, thielavin B, tiflamizole, timegadine, tolectin, tolpadol,tryptamid and those designated by company code number such as 480156S,AA861, AD1590, AFP802, AFPS60, A177B, AP504, AU8001, BPPC, BW540C,CHINOIN 127, CN100, EB382, EL508, F1044, FK-506, GV3658, ITF182,KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102,PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281,SY6001, TA60, TAI-901 (4-benzoyl-1-indancarboxylic acid), TVX2706,U60257, UR2301 and WY41770. Structurally related NSAIDs having similaranalgesic and anti-inflammatory properties to the NSAIDs are alsointended to be encompassed by this group.

In still another specific embodiment, the present invention is directedto the use of a huE3α polypeptide, or antagonist (including, but notlimited to, anti-huE3α selective binding agents) in combination(pretreatment, post-treatment or concurrent treatment) with any of oneor more corticosteroids, prodrug esters or pharmaceutically acceptablesalts thereof for the treatment of the diseases and disorders recitedherein, including acute and chronic inflammation such as rheumaticdiseases, graft versus host disease and multiple sclerosis.Corticosteroids, prodrug esters and pharmaceutically acceptable saltsthereof include hydrocortisone and compounds which are derived fromhydrocortisone, such as 21-acetoxypregnenolone, alclomerasone,algestone, amcinonide, beclomethasone, betamethasone, betamethasonevalerate, budesonide, chloroprednisone, clobetasol, clobetasolpropionate, clobetasone, clobetasone butyrate, clocortolone, cloprednol,corticosterone, cortisone, cortivazol, deflazacon, desonide,desoximerasone, dexamethasone, diflorasone, diflucortolone,difluprednate, enoxolone, fluazacort, flucloronide, flumethasone,flumethasone pivalate, flucinolone acetonide, flunisolide, fluocinonide,fluorocinolone acetonide, fluocortin butyl, fluocortolone, fluocortolonehexanoate, diflucortolone valerate, fluorometholone, fluperoloneacetate, fluprednidene acetate, fluprednisolone, flurandenolide,formocortal, halcinonide, halometasone, halopredone acetate,hydro-cortamate, hydrocortisone, hydrocortisone acetate, hydrocortisonebutyrate, hydrocortisone phosphate, hydrocortisone 21-sodium succinate,hydrocortisone tebutate, mazipredone, medrysone, meprednisone,methylprednisolone, mometasone furoate, paramethasone, prednicarbate,prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodiumphosphate, prednisolone sodium succinate, prednisolone sodium21-m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolonetebutate, prednisolone 21-trimethylacetate, prednisone, prednival,prednylidene, prednylidene 21-diethylaminoacetate, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide andtriamcinolone hexacetonide. Structurally related corticosteroids havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of an huE3α polypeptide, or antagonist (including, but not limitedto, anti-huE3α selective binding agents) in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or moreslow-acting antirheumatic drugs (SAARDs) or disease modifyingantirheumatic drugs (DMARDS), prodrug esters, or pharmaceuticallyacceptable salts thereof for the treatment of the diseases and disordersrecited herein, including acute and chronic inflammation such asrheumatic diseases, graft versus host disease and multiple sclerosis.SAARDs or DMARDS, prodrug esters and pharmaceutically acceptable saltsthereof comprise: allocupreide sodium, auranofin, aurothioglucose,aurothioglycanide, azathioprine, brequinar sodium, bucillamine, calcium3-aurothio-2-propanol-1-sulfonate, chlorambucil, chloroquine,clobuzarit, cuproxoline, cyclo-phosphamide, cyclosporin, dapsone,15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquinegold salt, gold sodium thiomalate, gold sodium thiosulfate),hydroxychloroquine, hydroxychloroquine sulfate, hydroxyurea, kebuzone,levamisole, lobenzarit, melittin, 6-mercaptopurine, methotrexate,mizoribine, mycophenolate mofetil, myoral, nitrogen mustard,D-penicillamine, pyridinol imidazoles such as SKNF86002 and SB203580,rapamycin, thiols, thymopoietin and vincristine. Structurally relatedSAARDs or DMARDs having similar analgesic and anti-inflammatoryproperties are also intended to be encompassed by this group.

In another specific embodiment, the present invention is directed to theuse of a huE3α polypeptide, or antagonist (including, but not limitedto, anti-huE3α selective binding agents) in combination (pretreatment,post-treatment, or concurrent treatment) with any of one or more COX2inhibitors, prodrug esters or pharmaceutically acceptable salts thereoffor the treatment of the diseases and disorders recited herein,including acute and chronic inflammation. Examples of COX2 inhibitors,prodrug esters or pharmaceutically acceptable salts thereof include, forexample, celecoxib. Structurally related COX2 inhibitors having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

In still another specific embodiment, the present invention is directedto the use of a huE3α polypeptide, or antagonist (including, but notlimited to, anti-huE3α selective binding agents) in combination(pretreatment, post-treatment, or concurrent treatment) with any of oneor more antimicrobials, prodrug esters or pharmaceutically acceptablesalts thereof for the treatment of the diseases and disorders recitedherein including cachexia, muscle wasting diseases and other catabolicdisorders. Antimicrobials include, for example, the broad classes ofpenicillins, cephalosporilns and other beta-lactams, aminoglycosides,azoles, quinolones, macrolides, rifamycins, tetracyclines, sulfonamides,lincosamides and polymyxins. The penicillins include, but are notlimited to penicillin G, penicillin V, methicillin, nafcillin,oxacillin, cloxacillin, dicloxacillin, floxacillin, ampicillin,ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate, hetacillin,cyclacillin, bacampicillin, carbenicillin, carbenicillin indanyl,ticarcillin, ticarcillin/clavulanate, azlocillin, mezlocillin,peperacillin, and mecillinam. The cephalosporins and other beta-lactamsinclude, but are not limited to cephalothin, cephapirin, cephalexin,cephradine, cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan,cefoxitin, ceruroxime, cefonicid, ceforadine, cefixime, cefotaxime,moxalactam, ceftizoxime, cetriaxone, cephoperazone, ceftazidime,imipenem and aztreonam. The aminoglycosides include, but are not limitedto streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycinand neomycin. The azoles include, but are not limited to fluconazole.The quinolones include, but are not limited to nalidixic acid,norfloxacin, enoxacin, ciprofloxacin, ofloxacin, sparfloxacin andtemafloxacin. The macrolides include, but are not limited toerythomycin, spiramycin and azithromycin. The rifamycins include, butare not limited to rifampin. The tetracyclines include, but are notlimited to spicycline, chlortetracycline, clomocycline, demeclocycline,deoxycycline, guamecycline, lymecycline, meclocycline, methacycline,minocycline, oxytetracycline, penimepicycline, pipacycline,rolitetracycline, sancycline, senociclin and tetracycline. Thesulfonamides include, but are not limited to sulfanilamide,sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole andco-trimoxazole (trimethoprim/sulfamethoxazole). The lincosamidesinclude, but are not limited to clindamycin and lincomycin. Thepolymyxins (polypeptides) include, but are not limited to polymyxin Band colistin.

Human E3α Compositions and Administration

Therapeutic compositions are within the scope of the present invention.Such compositions may comprise a therapeutically effective amount of ahuE3α polypeptide, including a fragment, variant, derivative, or one ormore selective binding agents which either inhibit or stimulate anactivity of huE3α in admixture with a pharmaceutically acceptable agentsuch as a pharmaceutically acceptable formulation agent; wherein huE3αrefers to the polypeptide sequence of huE3αI or huE3αII.

Human E3α pharmaceutical compositions typically include atherapeutically or prophylactically effective amount of huE3αpolypeptide, (an inhibitor of huE3α action) nucleic acid molecule orselective binding agent in a mixture with one or more pharmaceuticallyand physiologically acceptable formulation agents selected forsuitability with the mode of administration. Suitable formulationmaterials or pharmaceutically acceptable agents include, but are notlimited to, antioxidants, preservatives, coloring, flavoring anddiluting agents, emulsifying agents, suspending agents, solvents,fillers, bulking agents, buffers, delivery vehicles, diluents,excipients and/or pharmaceutical adjuvants. For example, a suitablevehicle or carrier may be water for injection, physiological salinesolution, or artificial cerebrospinal fluid, possibly supplemented withother materials common in compositions for parenteral administration.Neutral buffered saline or saline mixed with serum albumin are furtherexemplary vehicles. The term “pharmaceutically acceptable carrier” or“physiologically acceptable carrier” as used herein refers to one ormore formulation agents suitable for accomplishing or enhancing thedelivery of the huE3α polypeptide, nucleic acid molecule or selectivebinding agent as a pharmaceutical composition.

Acceptable formulation materials preferably are nontoxic to recipientsand are preferably inert at the dosages and concentrations employed. Thematerials may include buffers such as phosphate, citrate, or otherorganic acids; antioxidants such as ascorbic acid; low molecular weightpolypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as tween, pluronics, or polyethylene glycol (PEG).

Typically, a huE3α molecule pharmaceutical composition will beadministered in the form of a composition comprising a purifiedpolypeptide, in conjunction with one or more physiologically acceptableagents. It will be appreciated that when used herein, the term “huE3αmolecule pharmaceutical composition” also encompasses compositionscontaining a nucleic acid molecule or selective binding agent of thepresent invention.

Neutral buffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. Other standard pharmaceutically acceptable agentssuch as diluents and excipients may be included as desired. For example,the huE3α polypeptide product may be formulated as a lyophilizate usingappropriate excipients such as sucrose. Other exemplary pharmaceuticalcompositions comprise Tris buffer of about pH 7.0–8.5, or acetate bufferof about pH 4.0–5.5, which may further include sorbitol or a suitablesubstitute therefor.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. In addition, the compositionmay contain other formulation materials for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation.

Similarly, the composition may contain additional formulation materialsfor modifying or maintaining the rate of release of huE3α polypeptide,nucleic acid molecule or selective binding agent, or for promoting theabsorption or penetration of huE3α such molecules.

The huE3α molecule pharmaceutical compositions can be administeredparenterally. Alternatively, the compositions may be administeredthrough the digestive tract, such as orally, or by inhalation. Whenparenterally administered, the therapeutic compositions for use in thisinvention may be in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such pharmaceutically acceptablecompositions, with due regard to pH, isotonicity, stability and thelike, is within the skill of the art.

A particularly suitable vehicle for parenteral injection is steriledistilled water in which a huE3α polypeptide is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles or beads, or liposomes,that provides for the controlled or sustained release of the productwhich may then be delivered as a depot injection. Other suitable meansfor the introduction of the desired molecule include implantable drugdelivery devices.

The pharmaceutical compositions of the present invention may includeother components, for example parenterally acceptable preservatives,tonicity agents, cosolvents, wetting agents, complexing agents,buffering agents, antimicrobials, antioxidants and surfactants, as arewell known in the art. For example, suitable tonicity enhancing agentsinclude alkali metal halides (preferably sodium or potassium chloride),mannitol, sorbitol, and the like. Suitable preservatives include, butare not limited to, benzalkonium chloride, thimerosal, phenethylalcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, andthe like. Hydrogen peroxide may also be used as preservative. Suitablecosolvents are for example glycerin, propylene glycol and polyethyleneglycol. Suitable complexing agents are for example caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers can be conventional buffers such as borate, citrate, phosphate,bicarbonate, or Tris-HCl.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pHtypically within a pH range of from about 5 to about 8.

In one embodiment of the present invention, huE3α polypeptidecompositions may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's pharmaceutical sciences, 18^(th) edition, A. R. Gennaro,ed., Mack Publishing Company (1990)) in the form of a lyophilized cakeor an aqueous solution.

The optimal pharmaceutical formulation will be determined by one skilledin the art depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See for example,Remington's Pharmaceutical Sciences, pp. 1435–1712. Such compositionsmay influence the physical state, stability, rate of in vivo release,and rate of in vivo clearance of the present huE3α polypeptides.

An effective amount of a huE3α polypeptide composition to be employedtherapeutically will depend, for example, upon the therapeuticobjectives such as the indication for which the huE3α polypeptide isbeing used, the route of administration, and the condition of thepatient. Accordingly, the clinician may titer the dosage and modify theroute of administration to obtain the optimal therapeutic effect. Atypical dosage may range from about 0.1 μg/kg to up to about 100 mg/kgor more, depending on the factors mentioned above. In other embodiments,the dosage may range from 1 μg/kg up to about 100 mg/kg; or 5 μg/kg upto about 100 mg/kg; or 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up toabout 100 mg/kg.

Typically, a clinician will administer the composition until a dosage isreached that achieves the desired effect. The composition may thereforebe administered as a single dose, or as two or more doses (which may ormay not contain the same amount of the desired molecule) over time, oras a continuous infusion via implantation device or catheter.

One skilled in the art will appreciate that the appropriate dosagelevels for treatment will thus vary depending, in part, upon themolecule delivered, the therapeutic context, type of disorder undertreatment, the age, and general health of the recipient.

The huE3α molecule pharmaceutical composition to be used for in vivoadministration typically must be sterile. This maybe accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using these methods may be conductedeither prior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration may be stored in lyophilizedform or in solution. In addition, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the moleculeis being used, the route of administration, and the size (body weight,body surface or organ size) and condition (the age and general health)of the patient. Accordingly, the clinician may titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 mg/kg to up to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 0.1 mg/kg up to about 100 mg/kg;or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the molecule in the formulation used. Typically, a clinician willadminister the composition until a dosage is reached that achieves thedesired effect. The composition may therefore be administered as asingle dose, or as two or more doses (which may or may not contain thesame amount of the desired molecule) over time, or as a continuousinfusion via implantation device or catheter.

Pharmaceutical compositions such as (1) slow-release formulations, (2)inhalant mists, or (3) orally active formulations are also envisioned.The huE3α molecule pharmaceutical composition generally is formulatedfor parenteral administration. Such parenterally administeredtherapeutic compositions are typically in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising the desired huE3αmolecule in a pharmaceutically acceptable vehicle. The huE3α moleculepharmaceutical compositions also may include particulate preparations ofpolymeric compounds such as polylactic acid, polyglycolic acid, etc. orthe introduction of the molecule into liposomes. Hyaluronic acid mayalso be used, and this may have the effect of promoting sustainedduration in the circulation.

In one embodiment, a pharmaceutical composition may be formulated forinhalation. For example, huE3α polypeptide may be formulated as a drypowder for inhalation. Human E3α polypeptide or nucleic acid moleculeinhalation solutions may also be formulated in a liquefied propellantfor aerosol delivery, with or without a liquified propellant. In yetanother embodiment, solutions may be nebulized. Pulmonary administrationis further described in PCT WO94/20069, which describes pulmonarydelivery of chemically modified proteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, huE3α polypeptideswhich are administered in this fashion can be formulated with or withoutthose carriers customarily used in the compounding of solid dosage formssuch as tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the huE3α polypeptide. Diluents, flavorings,low melting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

Another pharmaceutical composition may involve an effective quantity ofhuE3α polypeptides in a mixture with non-toxic excipients which aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or other appropriate vehicle, solutions can be preparedin unit dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional huE3α molecule formulations will be evident to those skilledin the art, including formulations involving huE3α molecules incombination with one or more other therapeutic agents. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. Seefor example, PCT/US93/00829 which describes controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions.

Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22: 547–556, 1983),poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.Res., 15: 167–27, 19811; and Langer, Chem. Tech., 12: 98–105, 1982),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also may include liposomes, which can be prepared by any ofseveral methods known in the art. (See e.g., Eppstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688–3692, 1985; EP 36,676; EP 88,046; EP143,949.)

Regardless of the manner of administration, the specific dose may becalculated according to body weight, body surface area or organ size.Further refinement of the appropriate dosage is routinely made by thoseof ordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. oral, inhalation, injection or infusionby intravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes, or by sustained release systems orimplantation device. Where desired, the compositions may be administeredcontinuously by infusion, by bolus injection devices or by implantationdevice.

Alternatively or additionally, the composition may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which the desired molecule has beenabsorbed or encapsulated. Where an implantation device is used, thedevice may be implanted into any suitable tissue or organ, and deliveryof the desired molecule may be directly through the device viadiffusion, time-released bolus, or via continuous administration, or viacatheter using continuous infusion.

It will further be appreciated that the huE3α polypeptides, includingfragments, variants, and derivatives, may be employed alone, together,or in combination with other polypeptides and pharmaceuticalcompositions. For example, the huE3α polypeptides may be used incombination with cytokines, growth factors, antibiotics,anti-inflammatories, and/or chemotherapeutic agents as is appropriatefor the indication being treated.

In some cases, it may be desirable to use huE3α pharmaceuticalcompositions in an ex vivo manner. In such instances, cells, tissues, ororgans that have been removed from the patient are exposed to huE3αpharmaceuticalcompositions after which the cells, tissues and/or organsare subsequently implanted back into the patient.

In other cases, a huE3α polypeptide can be delivered by implantingcertain cells that have been genetically engineered, using methods suchas those described herein, to express and secrete the polypeptides. Suchcells may be animal or human cells, and may be autologous, heterologous,or xenogeneic. Optionally, the cells may be immortalized. However, inorder to decrease the chance of an immunological response, the cellsmaybe encapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

Additional embodiments of the present invention relate to cells andmethods (e.g., homologous recombination and/or other recombinantproduction methods) for both the iii vitro production of therapeuticpolypeptides by means of homologous recombination and for the productionand delivery of therapeutic polypeptides by gene therapy or celltherapy.

It is further envisioned that huE3α polypeptides can be produced byhomologous recombination, or with recombinant production methodsutilizing control elements introduced into cells already containing DNAencoding huE3α polypeptides. For example, homologous recombinationmethods may be used to modify a cell that contains a normallytranscriptionally silent huE3α gene, or an under expressed gene, andthereby produce a cell which expresses therapeutically efficaciousamounts of huE3α polypeptides. Homologous recombination is a techniqueoriginally developed for targeting genes to induce or correct mutationsin transcriptionally active genes. Kucherlapati, Prog. in Nucl. AcidRes. & Mol. Biol., 36:301, 1989. The basic technique was developed as amethod for introducing specific mutations into specific regions of themammalian genome (Thomas et. al., Cell, 44: 419–428, 1986; Thomas andCapecchi, Cell, 51:503–512, 1987; Doetschman et al., Proc. Natl. Acad.Sci., 85: 8583–8587, 1988) or to correct specific mutations withindefective genes (Doetschman et al., Nature, 330: 576–578, 1987).Exemplary homologous recombination techniques are described in U.S. Pat.No. 5,272,071 (EP 9193051, EP Publication No. 505500; PCT/US90/07642,International Publication No. WO 91/09955).

Through homologous recombination, the DNA sequence to be inserted intothe genome can be directed to a specific region of the gene of interestby attaching it to targeting DNA. The targeting DNA is a nucleotidesequence that is complementary (homologous) to a region of the genomicDNA. Small pieces of targeting DNA that are complementary to a specificregion of the genome are put in contact with the parental strand duringthe DNA replication process. It is a general property of DNA that hasbeen inserted into a cell to hybridize, and therefore, recombine withother pieces of endogenous DNA through shared homologous regions. Ifthis complementary strand is attached to an oligonucleotide thatcontains a mutation or a different sequence or an additional nucleotide,it too is incorporated into the newly synthesized strand as a result ofthe recombination. As a result of the proofreading function, it ispossible for the new sequence of DNA to serve as the template. Thus, thetransferred DNA is incorporated into the genome.

Attached to these pieces of targeting DNA are regions of DNA which mayinteract with or control the expression of a huE3α polypeptide, e.g.,flanking sequences. For example, a promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredhuE3α polypeptide. The control element controls a portion of the DNApresent in the host cell genome. Thus, the expression of huE3αpolypeptide may be achieved not by transfection of DNA that encodes thehuE3α gene itself, but rather by the use of targeting DNA (containingregions of homology with the endogenous gene of interest) coupled withDNA regulatory segments that provide the endogenous gene sequence withrecognizable signals for transcription of a huE3α polypeptide.

In an exemplary method, the expression of a desired targeted gene in acell (i.e., a desired endogenous cellular gene) is altered by theintroduction, by homologous recombination into the cellular genome at apreselected site, by the introduction of DNA which includes at least aregulatory sequence, an exon and a splice donor site. These componentsare introduced into the chromosomal (genomic) DNA in such a manner thatthis, in effect, results in the production of a new transcription unit(in which the regulatory sequence, the exon and the splice donor sitepresent in the DNA construct are operatively linked to the endogenousgene). As a result of the introduction of these components into thechromosomal DNA, the expression of the desired endogenous gene isaltered.

Altered gene expression, as described herein, encompasses activating (orcausing to be expressed) a gene which is normally silent (unexpressed)in the cell as obtained, as well as increasing the expression of a genewhich is not expressed at physiologically significant levels in the cellas obtained. The embodiments further encompass changing the pattern ofregulation or induction such that it is different from the pattern ofregulation or induction that occurs in the cell as obtained, andreducing (including eliminating) the expression of a gene which isexpressed in the cell as obtained.

One method by which homologous recombination can be used to increase, orcause, huE3α polypeptide production from a cell's endogenous huE3α geneinvolves first using homologous recombination to place a recombinationsequence from a site-specific recombination system (e.g., Cre/loxP,FLP/FRT) (Sauer, Current Opinion In Biotechnology, 5: 521–527, 1994;Sauer, Methods In Enzymology, 225: 890–900, 1993) upstream (that is, 5′to) of the cell's endogenous genomic huE3α coding region. A plasmidcontaining a recombination site homologous to the site that was placedjust upstream of the genomic huE3α coding region is introduced into themodified cell line along with the appropriate recombinase enzyme. Thisrecombinase causes the plasmid to integrate, via the plasmid'srecombination site, into the recombination site located just upstream ofthe genomic huE3α coding region in the cell line (Baubonis and Sauer,Nucleic Acids Res., 21: 2025–2029, 1993; O'Gorman et al., Science, 251:1351–1355, 1991). Any flanking sequences known to increase transcription(e.g., enhancer/promoter, intron, translational enhancer), if properlypositioned in this plasmid, would integrate in such a manner as tocreate a new or modified transcriptional unit resulting in de novo orincreased huE3α polypeptide production from the cell's endogenous huE3αgene.

A further method to use the cell line in which the site specificrecombination sequence had been placed just upstream of the cell'sendogenous genomic huE3α coding region is to use homologousrecombination to introduce a second recombination site elsewhere in thecell line's genome. The appropriate recombinase enzyme is thenintroduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, translocation) (Sauer, CurrentOpinion In Biotechnology, 5: 521–527, 1994; Sauer, Methods InEnzymology, 225: 890–900, 1993) that would create a new or modifiedtranscriptional unit resulting in de novo or increased huE3α polypeptideproduction from the cell's endogenous huE3α gene.

An additional approach for increasing, or causing, the expression ofhuE3α polypeptide from a cell's endogenous huE3α gene involvesincreasing, or causing, the expression of a gene or genes (e.g.,transcription factors) and/or decreasing the expression of a gene orgenes (e.g., transcriptional repressors) in a manner which results in denovo or increased huE3α polypeptide production from the cell'sendogenous huE3α gene. This method includes the introduction of anon-naturally occurring polypeptide (e.g., a polypeptide comprising asite specific DNA binding domain fused to a transcriptional factordomain) into the cell such that de novo or increased huE3α polypeptideproduction from the cell's endogenous huE3α gene results.

The present invention further relates to DNA constructs useful in themethod of altering expression of a target gene. In certain embodiments,the exemplary DNA constructs comprise: (a) one or more targetingsequences; (b) a regulatory sequence; (c) an exon; and (d) an unpairedsplice-donor site. The targeting sequence in the DNA construct directsthe integration of elements (a)–(d) into a target gene in a cell suchthat the elements (b)–(d) are operatively linked to sequences of theendogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequences, (b) a regulatorysequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)–(f) such that the elements of (b)–(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon.

If the sequence of a particular gene is known, such as the nucleic acidsequence encoding a huE3α polypeptide presented herein, a piece of DNAthat is complementary to a selected region of the gene can besynthesized or otherwise obtained, such as by appropriate restriction ofthe native DNA at specific recognition sites bounding the region ofinterest. This piece serves as a targeting sequence upon insertion intothe cell and will hybridize to its homologous region within the genome.If this hybridization occurs during DNA replication, this piece of DNA,and any additional sequence attached thereto, will act as an Okazakifragment and will be incorporated into the newly synthesized daughterstrand of DNA. The present invention, therefore, includes nucleotidesencoding a huE3α polypeptide, which nucleotides may be used as targetingsequences.

Human E3α polypeptide cell therapy, e.g., the implantation of cellsproducing huE3α polypeptides, is also contemplated. This embodimentinvolves implanting cells capable of synthesizing and secreting abiologically active form of huE3α polypeptide. Such huE3αpolypeptide-producing cells can be cells that are natural producers ofhuE3α polypeptides or may be recombinant cells whose ability to producehuE3α polypeptides has been augmented by transformation with a geneencoding the desired huE3α polypeptide or with a gene augmenting theexpression of huE3α polypeptide. Such a modification may be accomplishedby means of a vector suitable for delivering the gene as well aspromoting its expression and secretion. In order to minimize a potentialimmunological reaction in patients being administered a huE3αpolypeptide, as may occur with the administration of a polypeptide of aforeign species, it is preferred that the natural cells producing huE3αpolypeptide be of human origin and produce huE3α polypeptide. Likewise,it is preferred that the recombinant cells producing huE3α polypeptidebe transformed with an expression vector containing a gene encoding ahuman huE3α polypeptide.

Implanted cells may be encapsulated to avoid the infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow the release of huE3α polypeptide, but that preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue. Alternatively, thepatient's own cells, transformed to produce huE3α polypeptides ex vivo,may be implanted directly into the patient without such encapsulation.

Techniques for the encapsulation of living cells are known in the art,and the preparation of the encapsulated cells and their implantation inpatients may be routinely accomplished. For example, Baetge et al.(WO95/05452; PCT/US94/09299) describe membrane capsules containinggenetically engineered cells for the effective delivery of biologicallyactive molecules. The capsules are biocompatible and are easilyretrievable. The capsules encapsulate cells transfected with recombinantDNA molecules comprising DNA sequences coding for biologically activemolecules operatively linked to promoters that are not subject to downregulation in vivo upon implantation into a mammalian host. The devicesprovide for the delivery of the molecules from living cells to specificsites within a recipient. In addition, see U.S. Pat. Nos. 4,892,538,5,011,472, and 5,106,627. A system for encapsulating living cells isdescribed in PCT Application WO91/10425 of Aebischer et al. See also,PCT Application WO91/10470 of Aebischer et al., Winn et al. Exper.Neurol., 113: 322–329, 1991, Aebischer et al., Exper. Neurol., 111:269–275, 1991; and Tresco et al., ASAIO, 38: 17–23, 1992.

In vivo and in vitro gene therapy delivery of huE3α polypeptides is alsoenvisioned. In vivo gene therapy may be accomplished by introducing thegene encoding huE3α polypeptide into cells via local injection of ahuE3α nucleic acid molecule or by other appropriate viral or non-viraldelivery vectors (Hefti, Neurobiology, 25: 1418–1435, 1994). Forexample, a nucleic acid molecule encoding a huE3α polypeptide may becontained in an adeno-associated virus vector for delivery to thetargeted cells (e.g., Johnson, International Publication No. WO95/34670;International Application No. PCT/US95/07178). The recombinantadeno-associated virus (AAV) genome typically contains AAV invertedterminal repeats flanking a DNA sequence encoding a huE3α polypeptideoperably linked to functional promoter and polyadenylation sequences.

Alternative suitable viral vectors include, but are not limited to,retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitisvirus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus,rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Pat. No.5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. No. 5,631,236 involving adenoviralvectors; U.S. Pat. No. 5,672,510 involving retroviral vectors; and U.S.Pat. No. 5,635,399 involving retroviral vectors expressing cytokines.

Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Gene therapy materials and methods may also include the useof inducible promoters, tissue-specific enhancer-promoters, DNAsequences designed for site-specific integration, DNA sequences capableof providing a selective advantage over the parent cell, labels toidentify transformed cells, negative selection systems and expressioncontrol systems (safety measures), cell-specific binding agents (forcell targeting), cell-specific internalization factors, andtranscription factors to enhance expression by a vector as well asmethods of vector manufacture. Such additional methods and materials forthe practice of gene therapy techniques are described in U.S. Pat. No.4,970,154 involving electroporation techniques; WO96/40958 involvingnuclear ligands; U.S. Pat. No. 5,679,559 describing alipoprotein-containing system for gene delivery; U.S. Pat. No. 5,676,954involving liposome carriers; U.S. Pat. No. 5,593,875 concerning methodsfor calcium phosphate transfection; and U.S. Pat. No. 4,945,050 whereinbiologically active particles are propelled at cells at a speed wherebythe particles penetrate the surface of the cells and become incorporatedinto the interior of the cells.

In yet other embodiments, regulatory elements can be included for thecontrolled expression of the huE3α gene in the target cell. Suchelements are turned on in response to an appropriate effector. In thisway, a therapeutic polypeptide can be expressed when desired. Oneconventional control means involves the use of small molecule dimerizersor rapalogs (as described in WO9641865 (PCT/US96/099486); WO973 1898(PCT/US97/03137) and WO9731899 (PCT/US95/03157)) used to dimerizechimeric proteins which contain a small molecule-binding domain and adomain capable of initiating biological process, such as a DNA-bindingprotein or transcriptional activation protein. The dimerization of theproteins can be used to initiate transcription of the huE3α gene.

Other suitable control means or gene switches include, but are notlimited to, the following systems. Mifepristone (RU486) is used as aprogesterone antagonist. The binding of a modified progesterone receptorligand-binding domain to the progesterone antagonist activatestranscription by forming a dimer of two transcription factors which thenpass into the nucleus to bind DNA. The ligand binding domain is modifiedto eliminate the ability of the receptor to bind to the natural ligand.The modified steroid hormone receptor system is further described inU.S. Pat. No. 5,364,791; WO9640911, and WO9710337.

Yet another control system uses ecdysone (a fruit fly steroid hormone)which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a transactivation domain/DNA-bindingdomain/ligand-binding domain to initiate transcription. The ecdysonesystem is further described in U.S. Pat. No. 5,514,578; WO9738117;WO9637609; and WO9303162.

Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758; 5,650,298 and 5,654,168.

Additional expression control systems and nucleic acid constructs aredescribed in U.S. Pat. Nos. 5,741,679 and 5,834,186 to InnovirLaboratories Inc.

One example of a gene therapy technique is to use the huE3α gene (eithergenomic DNA, cDNA, and/or synthetic DNA encoding a huE3α polypeptidewhich may be operably linked to a constitutive or inducible promoter toform a “gene therapy DNA construct”. The promoter may be homologous orheterologous to the endogenous huE3α gene, provided that it is active inthe cell or tissue type into which the construct will be inserted. Othercomponents of the gene therapy DNA construct may optionally include, DNAmolecules designed for site-specific integration (e.g., endogenoussequences useful for homologous recombination), tissue-specificpromoter, enhancer(s) or silencer(s), DNA molecules capable of providinga selective advantage over the parent cell, DNA molecules useful aslabels to identify transformed cells, negative selection systems, cellspecific binding agents (as, for example, for cell targeting),cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as factors to enable vectormanufacture.

This gene therapy DNA construct can then be introduced into cells(either ex vivo or in vivo). One means for introducing the gene therapyDNA construct is by means of viral vectors as described herein. Certainvectors, such as retroviral vectors, will deliver the gene therapy DNAconstruct to the chromosomal DNA of the cells, and the gene therapy DNAconstruct can integrate into the chromosomal DNA. Other vectors willfunction as episomes, and the gene therapy DNA construct will remain inthe cytoplasm.

Another means to increase endogenous huE3α polypeptide expression in acell via gene therapy is to insert one or more enhancer elements intothe huE3α polypeptide promoter, where the enhancer element(s) can serveto increase transcriptional activity of the huE3α gene. The enhancerelement(s) used will be selected based on the tissue in which onedesires to activate the gene(s); enhancer elements known to conferpromoter activation in that tissue will be selected. For example, if agene encoding a huE3α polypeptide is to be “turned on” in T-cells, thelck promoter enhancer element may be used. Here, the functional portionof the transcriptional element to be added may be inserted into afragment of DNA containing the huE3α polypeptide promoter (andoptionally, inserted into a vector and/or 5′ and/or 3′ flankingsequence(s), etc.) using standard cloning techniques. This construct,known as a “homologous recombination construct”, can then be introducedinto the desired cells either ex vivo or in vivo.

Gene therapy can be used to decrease huE3α polypeptide expression bymodifying the nucleotide sequence of the endogenous promoter(s). Suchmodification is typically accomplished via homologous recombinationmethods. For example, a DNA molecule containing all or a portion of thepromoter of the huE3α gene(s) selected for inactivation can beengineered to remove and/or replace pieces of the promoter that regulatetranscription. For example the TATA box and/or the binding site of atranscriptional activator of the promoter may be deleted using standardmolecular biology techniques; such deletion can inhibit promoteractivity thereby repressing the transcription of the corresponding huE3αgene. The deletion of the TATA box or the transcription activatorbinding site in the promoter may be accomplished by generating a DNAconstruct comprising all or the relevant portion of the huE3αpolypeptide promoter(s) (from the same or a related species as the huE3αgene(s) to be regulated) in which one or more of the TATA box and/ortranscriptional activator binding site nucleotides are mutated viasubstitution, deletion and/or insertion of one or more nucleotides. As aresult, the TATA box and/or activator binding site has decreasedactivity or is rendered completely inactive. This construct, which alsowill typically contain at least about 500 bases of DNA that correspondto the native (endogenous) 5′ and 3′ DNA sequences adjacent to thepromoter segment that has been modified, may be introduced into theappropriate cells (either ex vivo or in vivo) either directly or via aviral vector as described herein. Typically, the integration of theconstruct into the genomic DNA of the cells will be via homologousrecombination, where the 5′ and 3′ DNA sequences in the promoterconstruct can serve to help integrate the modified promoter region viahybridization to the endogenous chromosomal DNA.

Other gene therapy methods may also be employed where it is desirable toinhibit the activity of one or more huE3α polypeptides. For example,antisense DNA or RNA molecules, which have a sequence that iscomplementary to at least a portion of the selected huE3α gene(s) can beintroduced into the cell. Typically, each such antisense molecule willbe complementary to the start site (5′ end) of each selected huE3α gene.When the antisense molecule then hybridizes to the corresponding huE3αmRNA, translation of this mRNA is prevented or reduced. It will also beappreciated by those skilled in the art that antisense and ribozymemolecules may also be administered directly.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more huE3α polypeptides. In thissituation, the DNA encoding a mutant full length or truncatedpolypeptide of each selected huE3α polypeptide can be prepared andintroduced into the cells of a patient using either viral or non-viralmethods as described herein. Each such mutant is typically designed tocompete with endogenous polypeptide in its biological role.

Additional Uses of huE3α Nucleic Acids and Polypeptides

Nucleic acid molecules of the present invention may be used to map thelocations of the huE3α gene and related genes on chromosomes. Mappingmay be done by techniques known in the art, such as PCR amplificationand in situ hybridization.

The nucleic acid molecules are also used as antisense inhibitors ofhuE3α polypeptide expression. Such inhibition may be effected by nucleicacid molecules which are complementary to and hybridize to expressioncontrol sequences (triple helix formation) or to huE3α mRNA. Antisenseprobes may be designed by available techniques using the sequence ofhuE3α nucleic acid molecules disclosed herein. Antisense inhibitorsprovide information relating to the decrease or absence of a huE3αpolypeptide in a cell or organism.

Hybridization probes may be prepared using the huE3α nucleic acidsequences provided herein to screen cDNA, genomic or synthetic DNAlibraries for related sequences. Regions of the DNA and/or amino acidsequence of huE3α polypeptide that exhibit significant identity to knownsequences are readily determined using sequence alignment algorithms asdescribed herein and those regions maybe used to design probes forscreening.

Human E3α nucleic acid molecules, as well as fragments, variants, and/orderivatives that do not themselves encode biologically activepolypeptides, may be useful as hybridization probes in diagnostic assaysto test, either qualitatively or quantitatively, for the presence ofhuE3α DNA or corresponding RNA in mammalian tissue or bodily fluidsamples.

Human E3α polypeptide fragments, variants, and/or derivatives, whetherbiologically active or not, are also useful for preparing antibodiesthat bind to a huE3α polypeptide. The antibodies may be used for invitro diagnostic purposes, including, but not limited to, use in labeledform to detect the presence of huE3α polypeptide in a body fluid or cellsample.

The full length cDNAs encoding huE3αI was subcloned into pCR 2.1 vector(Invitrogen, Cat.# K2030-40). The full length cDNA encoding huE3αII wassubcloned into pcDNA 3.1/His A vector (Invitrogen Cat.#V38-20). The fulllength cDNA encoding muE3 αII was subcloned into pCR 2.1 vector(Invitrogen). The above plasmids were deposited on Mar. 15, 2000 to theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. The plasmid containing huE3α1 is designated PTA-1489,the plasmid containing huE3αII is designated PTA-1490 and the plasmidcontaining muE3αII is designated PTA-1488.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLE 1 Cloning of cDNA Encoding Human E3αI

Materials and methods for cDNA cloning and analysis are described inSambrook et al., supra, which is incorporated herein by reference.

BLAST analysis of the Genebank dbEST database with the full lengthmurine E3α ubiquitin ligase nucleotide sequence (muE3I; GenebankAccession No.: AF061555; SEQ ID NO: 15), revealed 4 human EST sequences(Genebank accession numbers AI187306, AI92195, AI87306, and AI400279)which potentially encode different regions of a novel human E3αubiquitin ligase ortholog (huE3αI) gene. Based on these EST sequences,two sets of PCR primers (#2282-91/2282-93 and #2282-94/2282-97) weredesigned. These sequences are set out below in Table III.

TABLE III Primer Sequence SEQ ID NO: 2282-91 CTC CTC GAG TCT GCG TCA AAC7 2385-35 TCT GCA TAT GTT CAG CCT TGC TA 8 2282-94 GTA TGA ACT TGC CGAGGC TTT TA 9 2294-37 CAA TAC TTT CCC AGC CCT CAG AA 10

The primer sets #2282-91/2282-93 (SEQ ID NOS: 7 and 8) and#2282-94/2294-37 (SEQ ID NOS: 9 and 10) were used to generate two PCRproducts which span the whole huE3αI gene including the 5′ and 3′untranslated regions. Polymerase chain reactions (PCR) were performedusing a Perkin-Elmer 9600 thermocycler. In general, 50 μl PCR reactionscontained 24 μl of H₂O, 5 μl of 10×cDNA PCR Reaction Buffer (Clontech),2 μl of 10 mM dNTP mix (dATP, dCTP, dGTP, dTTP), 1 μl of Primer 2282-91or 2282-94 (20 μl), 1 μl of Primer 2285-35 or 2294-37 (20 μl), 2 μl of50× Advantage 2 Polymerase Mix (Clontech) and 15 μl of Marathon ReadycDNA from a human heart library (Clontech cat.# 7404-1) or a humanmuscle library (Clontech cat. # 7413-1). The reaction mixture wasincubated at 94° C. for 30 seconds, followed by 40 cycles of 94° C. for30 seconds, 60° C. for 30 seconds, and 72° C. for 5 minutes.

The PCR products were electrophoresed on a 1% agarose gel as describedby Sambrook et al., supra. The appropriate sized bands (14 kB and 3 kB)were excised from the agarose gel and purified with the QIAquick GelExtraction kit (Qiagen, cat# 28704). The two purified DNA fragments weresubcloned into pCR2.1 vectors and transformed into E. coli (StrainINVαF) utilizing the Invitrogen Original TA Cloning kit (cat.#K2000-40).

After subcloning, DNA plasmids were purified with the QIAprep SpinMiniprep kit (cat# 27104). The sequence of the PCR products wereverified by automated sequencing with the Prism 377 Sequencer and theBIg Dye Terminator Ready Reaction mix with AmpliTaq DNA polymerase(Perkin Elmer Applied Biosystems). Each sequencing reaction wasperformed in a Perkin Elmer 9600 thermocycler with 25 cycles of 96° C.for 10 seconds, 50° C. for 5 seconds and 60° C. for 2 minutes. Thesamples were purified using Centriflex Gel Filtration cartridges (EdgeBiosystems). The samples were heated to 85° C. for 2 minutes andinserted into the Prism 377 Sequencer. The sequences were analyzed usingthe Sequnecher™ Sequence Analysis software (Gene Codes Corp.). Thesequences of the PCR product generated from human heart and human musclewere identical. The full length huE3αI clone was obtained by ligatingthe two PCR products together at their XbaI site.

The nucleic acid sequence of huE3αI (SEQ ID NO: 1), consists of an openreading frame of 5247 nucleotides which encodes a 1749 amino acidpolypeptide, in addition to 695 bp in the 5′ untranslated region and 362bp in the 3′ untranslated region. Alignment of the human and mouse aminoacid sequence, as shown in FIG. 1 (SEQ ID NOS: 2 and 15, respectively),exhibited 92.5% overall sequence identity.

In the present invention, a novel full length human E3α cDNA (huE3αI;SEQ ID NO: 1) was isolated and cloned and the full length polypeptidesequence (SEQ ID NO: 2) was disclosed. A partial sequence of the humanE3α gene had been previously reported. (See U.S. Pat. No. 5,861,312;Kwon et al., Proc. Natl. Acad. of Sci. USA, 95: 7898–7903, 1998). Thereported partial sequence is encompassed in SEQ ID NO: 1; but onlyrepresents a small portion of the entire full length gene (nucleotides702 to 1066).

EXAMPLE 2 Cloning of cDNA Encoding Human E3α Ortholog, huE3αII

BLAST analysis of the Amgenesis database (Amgen internal EST database)with the human E3αI amino acid sequences revealed 4 Amgenesis ESTsequences (amgi-039645, smop2-0079fl2 and zhgb-aa693825 and Genebankaccession no.: AA002347) which encode potential regions of the human andmouse E3α ubiquitin ligase ortholog nucleotide sequences which aredeonted as E3αII. Based on the zhgb-aa693825 and AA002347 sequences, twoPCR primer sets (#2380-88/2378-32 and #2381-48/2385-94) were designed.These sequences are set out below in Table IV.

TABLE IV Primer Sequence SEQ ID NO: 2380-88 ATG GCG TCG CTA GAG CCA 112378-32 CAA AGC GGC TGA GCA TGA TCA TC 12 2381-48 TGA ACA GCC AAT CACACT AAG CA 13 2385-94 TTA TAA ATG CCA GTC AAT GCC AA 14

The primer sets #2380-88/2378-32 (SEQ ID NOS: 11 and 12) and#2381-48/2385-94 (SEQ ID NOS: 13 and 14) were used to generate two PCRproducts which encode the coding region of a novel ortholog of human E3αligase (huE3αII). The 5′ and 3′ untranslated regions of huE3αII weredetermined based on the EST sequences amgi-03645 and smop2-0079fl2 inorder to obtain the full length huE3αII cDNA. PCR was performed asdescribed above utilizing Marathon-Ready cDNA from human heart and humanmuscle libraries. The two PCR products were electrophoresed on a 1% gelas described by Sambrook et al., supra. The appropriate sized bands (2.2kB and 3.5 kB) were excised from the agarose and purified by QIAquickGel Extraction kit (cat.# 28704). The PCR products were subcloned intothe pcDNA3.1-HisA vector (Invitrogen cat.# V385-20) and transformed intoE. coli (Strain INVαF) using the Invitrogen Original TA Cloning kit. Theinsert DNA was purified with the QIAprep Spin Miniprep kit (QIAGEN cat.#27104) and subsequenly digested with NotI/SacI for the 2 kB product andSacI/XhoI for the 3.3 kB product. The PCR products were sequenced asdescribed in Example 1 and the products generated from human heart andhuman muscle cDNA libraries were identical. The full length huE3αII genewas generated by ligating these two PCR products at their SacI sites.

The nucleic acid sequence of huE3αII (SEQ ID NO: 3), consists of an openreading frame of 5265 nucleotides which encodes a 1755 amino acidpolypeptide, in addition to 294 bp in the 5′ untranslated region and 740bp in the 3′ untranslated region. Alignment of the human and mouse aminoacid E3αII sequences, as shown in FIG. 1 (SEQ ID NOS: 4 and 6,respectively), exhibited 90.4% overall sequence identity. There is a48.1% overall amino acid sequence identity between human E3αI and humanE3αII (SEQ ID NOS:2 and 4, respectively).

In the present invention, a novel full length cDNA sequence encodinghuE3αII (SEQ ID NO: 3) was cloned and isolated, and the full lengthpolypeptide sequence was disclosed (SEQ ID NO: 4). A partial sequence ofhuE3αII was identified in WO9904265 as one of many partial sequenceswith unknown identities that were speculated to be cancer markers.

EXAMPLE 3 Cloning of the Murine E3αII Ortholog

BLAST analysis of the Amgen internal database, Amgensis, with humanE3αII amino acid sequences identified the mouse cDNA clone(Smop2-00079-fl2) as a potential mouse ortholog of E3αII ubiquitinligase. The Amgenesis database contained the entire coding region of themouse E3αII ubiquitin ligase (muE3αII) gene. The cDNA clone of wasobtained from the Amgen sequencing group. The sequence of the clone wasconfirmed to be the full cDNA of muE3αII as described in Example 1. Thenucleic acid sequence of muE3αII (SEQ ID NO: 5), consists of an openreading frame of 5265 nucleotides which encodes a 1755 amino acidpolypeptide, in addition to 765 bp in the 5′ untranslated region and 56bp in the 3′ untranslated region.

EXAMPLE 4 Human E3αII Tissue Expression

Tissue expression patterns of huE3αI and huE3αII mRNA were analyzed byNorthern blot analysis. To detect the presence of huE3αII transcript invarious tissues, a ³²P-labeled fragment of huE3αII, which was 452 bp andcorresponded to nucleotides 3557–4009 of SEQ ID NO: 3, was used as aprobe. For detection of huE3αI transcript in various tissues, a³²P-labeled fragment of huE3αI, which was 696 bp and corresponded tonucleotides 3468–4164 of SEQ ID NO: 1, was used as a probe. The probeswere labeled by random priming method using Prime-it RMT labeling kit(Stratagene, Cat# 300392). The specific activities was 1.436×10⁶ cpm/μlfor the huE3αII probe and 1.207×10⁶ cpm/μl for the huE3αI probe. Humanmultiple tissue poly A+ RNA blots (Clontech cat.# 7780-1) wereprehybridized in Church hybridization solution (1% BSA, 7% SDS, 0.5 Msodium phosphate, pH 7.0, 1 mM EDTA) for 4 hour at 65° C. The blots arethen hybridized in Church hybridization solution With 3.0×10⁶ cpm/ml ³²Plabeled probe for overnight at 65° C. The blots are then washed 3 timesin Wash B buffer (1% SDS, 0.04 M sodium phosphate, 1 mM EDTA) for 5minutes each at room temperature, followed by two times at 65° C. Theblots were exposed to X-ray film at room temperature overnight (forhuE3αII detection) or one week (for huE3αI detection).

The Northern blot analysis revealed that huE3αII (FIG. 2) ispredominantly expressed in skeletal muscle, with moderate expression inheart and kidney tissue and minimal or no expression in other tissuesexamined including brain, colon, thymus, spleen, liver, smallintestines, placenta, lung and peripheral white blood cells. Incontrast, the expression of huE3αI (FIG. 3) is less muscle-specific.Although heart and skeletal muscle had relative high levels of huE3αItranscripts, moderate levels of huE3αI was found to spread through thevarious tissues examined. The results indicate that huE3αII is the moremuscle-specific form of huE3α which is predominantly expressed inskeletal muscle tissue.

EXAMPLE 5 Production of huE3α Polypeptides

A. Bacterial Expression of huE3α Polypeptides

PCR is used to amplify template DNA sequences encoding a huE3αpolypeptide using primers corresponding to the 5′ and 3′ ends of thesequence. The amplified DNA products may be modified to containrestriction enzyme sites to allow for insertion into expression vectors.PCR products are gel purified and inserted into expression vectors usingstandard recombinant DNA methodology. An exemplary vector, such aspAMG21 (ATCC No. 98113) containing the lux promoter and a gene encodingkanamycin resistance is digested with BamHI and NdeI for directionalcloning of inserted DNA. The ligated mixture is transformed into an E.coli host strain by electroporation and transformants are selected forkanamycin resistance. Plasmid DNA from selected colonies is isolated andsubjected to DNA sequencing to confirm the presence of the insert.

Transformed host cells are incubated in 2×YT medium containing 30 μg/mlkanamycin at 30° C. prior to induction. Gene expression is induced bythe addition of N-(3-oxohexanoyl)-dl-homoserine lactone to a finalconcentration of 30 ng/ml followed by incubation at either 30° C. or 37°C. for six hours. The expression of huE3α polypeptide is evaluated bycentrifugation of the culture, resuspension and lysis of the bacterialpellets, and analysis of host cell proteins by SDS-polyacrylamide gelelectrophoresis.

Inclusion bodies containing huE3α polypeptide are purified as follows.Bacterial cells are pelleted by centrifugation and resuspended in water.The cell suspension is lysed by sonication and pelleted bycentrifugation at 195,000×g for 5 to 10 minutes. The supernatant isdiscarded, and the pellet is washed and transferred to a homogenizer.The pellet is homogenized in 5 ml of a Percoll solution (75% liquidPercoll/0.15 M NaCl) until uniformly suspended and then diluted andcentrifuged at 21,600×g for 30 minutes. Gradient fractions containingthe inclusion bodies are recovered and pooled. The isolated inclusionbodies are analyzed by SDS-PAGE. A single band on an SDS polyacrylamidegel corresponding to E. coli-produced huE3α polypeptide is excised fromthe gel, and the N-terminal amino acid sequence is determinedessentially as described by Matsudaira et al., J. Biol. Chem., 262:10–35 (1987).

B. Mammalian Cell Production of huE3α Polypeptides

The huE3α DNA was subcloned into a mammalian expression vector asdescribed above using standard DNA technology. An exemplary expressionvector, pCEP4 (Invitrogen, Carlsbad, Calif.), which contains anEpstein-Barr virus origin of replication, may be used for the expressionof huE3α in 293-EBNA-1 cells. Amplified and gel purified PCR productsare ligated into pCEP4 vector and lipofected into 293-EBNA cells. Thetransfected cells are selected in 100 μg/ml hygromycin and the resultingdrug-resistant cultures are grown to confluence. The cells are thencultured in serum-free media for 72 hours. The conditioned media isremoved and, huE3α protein polypeptide expression is analyzed bySDS-PAGE. Human E3α polypeptide expression may be detected by silverstaining. Alternatively, huE3α polypeptide is produced as a fusionprotein with an epitope tag, such as an IgG constant domain or a FLAGepitope, which may be detected by Western blot analysis using antibodiesto the tag peptide.

Human E3α polypeptides may be excised from an SDS-polyacrylamide gel, orhuE3α fusion proteins are purified by affinity chromatography to theepitope tag, and subjected to N-terminal amino acid sequence analysis asdescribed herein.

EXAMPLE 6 Production of Anti-huE3α Polypeptide Antibodies

Antibodies to huE3α polypeptides may be obtained by immunization withpurified protein or with huE3α peptides produced by biological orchemical synthesis. Suitable procedures for generating antibodiesinclude those described in Hudson and Bay, Practical Immunology, SecondEdition”, Edition, Blackwell Scientific Publications.

In one procedure for the production of antibodies, animals (typicallymice or rabbits) are injected with a huE3α antigen (such as anrecombinant truncated forms of huE3α polypeptide), and those withsufficient serum titer levels as determined by ELISA are selected forhybridoma production. Spleens of immunized animals are collected andprepared as single cell suspensions from which splenocytes arerecovered. The splenocytes are fused to mouse myeloma cells (such asSp2/0-Ag14 cells), allowed to incubate in DMEM with 200 U/ml penicillin,200 μg/ml streptomycin sulfate, and 4 mM glutamine, then incubated inHAT selection medium (Hypoxanthine; Aminopterin; Thymidine). Afterselection, the tissue culture supernatants are taken from each fusionwell and tested for anti-huE3α antibody production by ELISA.

Alternative procedures for obtaining anti-huE3α antibodies may also beemployed, such as the immunization of transgenic mice harboring human Igloci for production of human antibodies, and the screening of syntheticantibody libraries, such as those generated by mutagenesis of anantibody variable domain.

EXAMPLE 7 Biological Activity of huE3α Polypeptides

Human E3α family members are known to catalyze the ubiquitin conjugationreaction which ultimately results in protein degradation. To determinethe biological activity of huE3α polypeptide, the rate of ubiquitinconjugation and the rate of protein degradation are measured. Thefollowing are examples of assays to measure these biological activities.

A. Ubiquitin Conjugation Assay:

The enzymatic activity of E3α family members is thought to be the ratelimiting step in ubiquitin conjugation. Rat skeletal muscles aredissected, homogenized, and centrifuged at 100,000×g to removeproteosomes. The soluble extract is incubated with ¹²⁵I-ubiquitin(Amersham, Arlington Heights, Ill.) (0.15 mg/ml) in 20 mM Tris (pH 7.4),1 mM DTT, 5 mM MgCl₂, and 2 mM ATPγS at 37° C. in the presence andabsence of huE3α polypeptide. At various time points, the reactions areterminated by the addition of sample buffer and SDS-PAGE is performed ona 12% gel. The gel is then dried and autoradiographed. If huE3α acts asan E3α family member, the level of ubiquitination should increase inextracts treated with the huE3α polypeptide (Soloman et al., Proc. Natl.Acad. of Sci. U.S.A., 95: 12602–07, 1998).

B. Protein Degradation Assays:

Measurement of tyrosine release is a preferred method for determiningthe rate of protein turnover in skeletal muscles. Rat skeletal musclesare dissected and homogenated. The extracts are incubated at 37° C. for2 hours in 20 mM Tris (pH 7.6), 5 mM MgCl₂, 2 mM DTT, ATP-regeneratingsystem (10 μg creatine phosphokinase and 10 mM creatine phosphate), 1 mMATP, and 25 mg of ubiquitin in the presence and absence of huE3αpolypeptide. Subsequently, the reactions are terminated with 20% TCA.After centrifugation, the concentrations of tyrosine in the supernatantis measured by fluorescence spectroscopy according to the method ofWaakkes and Udenfriend (J. Lab. Clin. Med., 50: 733–736, 1957).

Measurement of radiolabeled proteins will also indicate if huE3αpolypeptide exhibits E3α family biological activity. Rat skeletal musclehomogenates are incubated at 37° C. for 2 hours with ¹²⁵I-labeled N-endpathway substrates, such as ¹²⁵I-lyzozyme and ¹²⁵I-lactalbumin, in thepresence and absence of huE3α polypeptide. Following the incubation, 20%TCA is added to precipitate the radioactivity. The release ofTCA-soluble radioactivity is measured using a gamma counter andcorrelates the rate of protein degradation. The addition of huE3αpolypeptide should increase the rate of protein degradation in both ofthese assays.

EXAMPLE 8 Identification of Modulators of the Biological Activity ofhuE3α Polypeptides

The assays described in Example 7 demonstrate preferred methods tomeasure the biological activity of huE3α as an ubiquitin ligase. Thesemethods are also useful for identifying modulators of huE3α ubiquitinligase activity.

The rate limiting step of ubiquitin conjugation consists of E3αcatalyzing the transfer of the activated ubiquitin molecule to thetarget protein. The rate of ubiquitination modulated by huE3α can bemeasured in dissected rat skeletal muscles as described in Example 7.The addition of potential huE3α modulators (inhibitors or stimulators)to this system will allow for the identification of E3α stimulators andinhibitors by virtue of their ability to modulate the level or rate ofubiquitin conjugation to the target protein. If the addition of themodulator decreases the rate of huE3α-modulated ubiquitin conjugation,it is considered a huE3α inhibitor. If the modulator increases the rateof ubiquitin conjugation it is considered a stimulator.

The effect of huE3α modulators can also be determined by measuring theireffect on the rate of protein turnover as described in Example 7. IfhuE3α exhibits the biological activity of an ubiquitin ligase, it willinduce protein degradation. Protein turnover is measured by quantitatingtyrosine release or the degradation of radioactively labeled N-endpathway substrates in the presence of E3α modulators. The addition ofeffective huE3α modulators will either increase or decrease the rate ofprotein degradation.

EXAMPLE 9 Identification of huE3αI Single Nucleotide Polymorphisms (SNP)

A BLAST search of the Celera Human Genome database was conducted usingthe huE3αI cDNA sequence (SEQ ID NO: 1) as a probe. The sequencesidentified in the search were used to manually assemble a polynucleotidesequence (SEQ ID NO: 18) which was discovered to have a singlenucleotide mismatch at nucleotide 5397 of the huE3αI cDNA sequence (SEQID NO: 1). The polynucleotide sequence of SEQ ID NO: 18 contians ahuE3αI SNP with a change of a cytosine to a thymidine at position 4702,which caused the predicted amino acid sequence of SEQ ID NO: 2 to changefrom an Arg residue to a W (Trp) residue at position 1508.

PCR was carried out to confirm the polynucleotide sequence of huE3αIcDNA. Primers were designed to flank the mismatch as follows: 5′AGAAGGAGAGTACAGTGCACTC3′ (SEQ ID NO: 20) and 5′CGAAAGCATCCTGTCCTCTG (SEQID NO: 21). PCR was carried out as described in Example 1 with theMarathon-Ready cDNA library (Clontech cat no. 7413-1) from which huE3αIcDNA was cloned. The PCR reactions resulted in 8 individual PCR productswhich had identical sequences to the huE3αI SNP (SEQ ID NO: 18).

These experiments have confirmed the sequence of a huE3α1 SNP set out inSEQ ID NO: 18 wherein the nucleotide at position 4657 is a cytosine.Accordingly, the correct predicted amino acid sequence is set out as SEQID NO: 19, wherein the residue at position 1573 is R (Arg).

EXAMPLE 10 Human E3αI and E3αII Stimulate Ubiquitination

To confirm that huE3αI and huE3αII have the predicted enzymatic activityof stimulating ubiquitin conjugation, ubiquitination reactions werecarried out in 293 cells. Cultures of 293T cells (ATCC accession no.CRL1573) were transfected with huE3αI or huE3αII full length cDNA (SEQID NOS: 1 or 3, respectively) that had been subcloned into pcDNA3.1vector (Invitrogen) under the control of the CMV promoter usingLipofectamine reagent 2000 (Gibco, cat no. 11668-027) according to themanufacture's instructions. As a control, 293T cultures were transfectedwith pcDNA3.1 vector without the cDNA insert. The transfected cells werelysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 8.0), 2 mM DTT, 5 mMMgCl₂) in the presence of Sigma P8340 protease inhibitor cocktail(containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64,bestatin, leupeptin and aprotinin) at 100 μl/10⁷ cells. The crudelysates were then centrifuged at 10,000 g for 10 minutes.

The supernatants prepared from vector-(Control), human E3α-I-(hu-E3α-I)or human E3α-II-(hu-E3α-II) transfected cells were subjected toubiquitination reactions. To measure ubiquitination of endogenousproteins, 30 μg of cell lysate was incubated with ¹²⁵I-ubquitin (0.15mg/ml, approximately 10⁷ cpm) in a total volume of 40 μl in a buffercontaining 50 mM Tris, pH 8.0, 2 mM DTT, 5 mM MgCl₂, 2 mM adenosine5′-[-thio]triphosphate (ATP S), 50 μg/ml ubiquitin aldehyde, MG132 20μg/ml and protease inhibitor cocktail (Sigma P8340) at 37° C. for 30minutes. Reactions were stopped by adding sample buffer and weresubjected to 12% SDS PAGE. The gels were then dried andautoradiographed.

The ubiquitination of α-lactalbumin, a known substrate for N-end RuleUbiquitination was also measured with the 239T transfected cells. Forthese reactions, 30 μg of cell lysate proteins was incubated with 0.15mg/ml ¹²⁵I-α-Lactalbumin and 0.25 mg/ml unlabeled ubiquitin in a totalvolume of 40 μl in a buffer containing 50 mM Tris (pH 8.0) 2 mM DTT, 5mM MgCl₂, 2 mM adenosine 5′-[-thio]triphosphate (ATP S), ubiquitinaldehyde 50 ug/ml, 20 μg/ml MG132 and protease inhibitor cocktail (SigmaP8340) at 37° C. for 30 minutes. Reactions were stopped by adding samplebuffer and each reaction was run of a 8% SDS PAGE was performed. Thegels were then dried and autoradiographed

The amount of radioactivity incorporated into high molecular weightbands denoted as “ubquitin-protein conjugates” in FIG. 4 (above 18 kDafor endogenous proteins and above 35 kDa for α-Lactalbumin) werequantitated by using PhosphaImager and plotted (right panel). Thesereactions indicated that recombinant expression of huE3αI or huE3 αII in293 cells lead to accelerated ubiquination of endogenous cellularproteins and ubiquitin conjugation to α-lactobumin, a bona fide N-endrule substrate.

To further substantiate the enzymatic activity of huE3αI and huE3αII,ubiquitin conjugation to endogenous cellular proteins were measured incultured muscle cell lines. Cultures of murine C₂C₁₂ or rat L6 myotubecells (ATCC accession nos. CRL-1772 and CRL-1458, respectively) weretransfected with huE3αI or huE3αII full length cDNA under control of theCMV promoter using Lipofectamine 2000 Reagent (Gibco). Mock transfectionwith the pcDNA3.1 vector without a cDNA insert was performed as acontrol. Cell lysates were prepared as described above for the 293Tcells and the resulting supernatants were used in ubiquitin conjugationreactions. For each reaction, 30 μg of C₂C₁₂ or L6 myotube cell lysatewas incubated with ¹²⁵I-ubiquitin (0.15 mg/ml, approximately ×10⁷ cpm)in a total volume of 25–30 μl in a buffer containing 50 mM Tris, pH 8.0,2 mM DTT, 5 mM MgCl2, 2 mM adenosine 5′-[-thio]triphosphate (ATP S), 50μg/ml ubiquitin aldehyde, 20 μg/ml MG132 and protease inhibitor cocktail(Sigma P8340) at 37° C. for 30 minutes. Reactions were stopped by addingsample buffer and were subjected to 12% SDS PAGE. The gels were thendried and autoradiographed.

The amount of ubiquitinated muscle proteins (¹²⁵I-Ubiquitin proteinconjugates) were quantitated as the total radioactivity incorporatedinto high molecular weight bands (above 18 kDa) using a Phsophoimager asshown in FIG. 5 (left panel). These reactions indicated thattransfection of huE3αI and huE3αII increased ubiquitination of cellularproteins 2–3 fold (see FIG. 5, right panel) in murine C₂C₁₂ and ratmyotube cultures.

EXAMPLE 11 Expression of Human E3αI and Human E3αII is UnregulatedDuring Cachexia Disease States

The Yoshida Hepatoma-130 (YAH) cachexia rat model as described inBaracos et al. (Am. J. Physiol., 268(5 Pt 1): E996–1006, 1995) was usedto determine if huE3αI and huE3αII are upregulated in cachexia diseasestates. For tumor implantation, female Sprague-Dawley rats of theBuffalo strain from a colony maintained at the University of Albertawere used as the host for the YAH tumor cells. Tumor cell stocks weremaintained in liquid nitrogen and used after two passages in recipientfemale animals of the same strain. Rats were housed in individual wiremesh cages in a temperature (24° C.)- and humidity (80%)-controlled roomon a 12:12-h light-dark cycle. Rats were fed ground laboratory chow(Continental Grain, Chicago, Ill.) containing 24% crude protein.

Rats were allocated by initial body weight to three groups such that thesizes (mean±SE) of the animals receiving each treatment were similar(˜200 g). Two different treatments were compared: YAH-bearing andpair-fed control rats. The pair-fed rats, which received one meal perevery day at 9.00 am, were fed on the basis of their body weights, thesame amount of food consumed by the tumor-bearing rats. On days 1, 2, 3,4 and 5 after tumor-implantation, food intake was determined inpreliminary experiments to be 9, 7.5, 5.3, 1.5, and 0.9% respectively,of initial body weight per day. Rats were implanted with 100 ml ofascites fluid containing YAH cells from a single donor animal. Thecontrol rats were implanted with an equal volume of saline buffer. Ratswere sacrificed by CO₂ asphyxiation after 3 and 5 days, andepitrochelaris, EDL, soleus, medial gastrocnemius muscles were rapidlydissected and the gastrocnemius muscles were weighed. Tissues werefrozen immediately in liquid nitrogen and stored at −70° C. until use.

The gastrocnemius skeletal muscle weights in YAH-130 tumor bearing ratswere significantly lower than those measured from the pair-fed controlrats. As indicated in the Table V below, the YAH-130 tumor bearing ratsunderwent muscle wasting by day 3 after tumor implantation which wasmore apparent at day 5 after implantation. The muscle weights arecalculated (in grams) as the mean±standard error.

Days after Tumor Pair Fed Control Tumor-Bearing Percent Implantation n(in grams) (in grams) Change 3 days 8 530 ± 14.6 508 ± 7.3  −4.3% 5 days8 593 ± 8.1  443 ± 9.4 −25.3% n = number of animals

The rate of ubiquitin conjugation of the endogenous muscle proteins werecarried out as described in Example 10 using the skeletal muscles fromthe YAH tumor-bearing rats. The frozen gastrocnemius muscles collected(via dissection at sacrifice) from 6 tumor-bearing rats were combined.The muscle extracts (20% weight/volume) were prepared by homogenizingthe muscles in a buffer containing 50 mM Tris HCl (pH 8.00), 5 mM MgCl₂,2 mM DTT, protease inhibitor cocktail (Sigma P8340) and 10% glycerol.The homogenates were then centrifuged at 40,000 g for 1 hr and theresulting supernatants were used as crude muscle extracts.

For some assays, the crude muscle extracts were fractionated further bychromatography on DEAE-cellulose (Whatman, Clifton, N.J.) to removeendogenous ubiquitin as described by Soloman et al. (Proc. natl. Acad.Sci. U.S.A., 95: 12602–7, 1998). The bound material Fraction II, whichcontained most of the ubiquitin conjugating enzymes were eluted with 50mM Tris, pH 8.0 containing 0.5M NaCl and 1 mM DTT. Both crude extractsand Fraction II were dialyzed prior to use for ubiquitination assayagainst buffer containing 20 mM Tris, pH 8.0, 2 mM DTT, 5 mM MgCl2, and10% glycerol and stored at 70° C. until use. Crude muscle extracts wereused for ubiquitin conjugation to 125I-α-lactalbumin. Fraction II wasused when rates of endogenous skeletal muscles proteins were comparedand also when effects of E3α inhibitors on skeletal muscle proteinubiquitination were tested.

The Fraction II from both tumor-bearing and pair-fed control rats weresubjected to ubiquitination reactions of the endogenous muscle proteinsas described in Example 10 in the presence of 20 μg/ml of bestatin and10 mM of either the E3α selective inhibitor arginine methyl ester(Arg-ME) or the control alanine methyl ester (Ala-ME) (Sigma Chemicals,St. Louis Mo.). The reactions were incubated at 37° C. for 20 minutesand the ¹²⁵I-Ubiquitin conjugates were resolved by 12% SDS PAGE asdescribed in Example 10.

As shown in FIG. 6, the tumor-bearing rats exhibited accelerated muscleprotein ubquitination. The increase in ubiquitination within the ratskeletal muscles of the tumor-bearing rats was attributable to theactivation of the E3α/N-end rule pathway, since the addition of E3αspecific inhibitor arginine methylester virtually abolished theaccelerated ubiquitination activity (see lanes 9 and 10 on FIG. 6).

To further establish the role of huE3αI and huE3αII in the N-end rulepathway in muscle wasting in the rat cachexia model, the rates ofubiquitination of N-end rule substrate α-lactalbumin was measured inskeletal muscle extracts from control and tumor-bearing mice.¹²⁵I-α-lactalbumin (0.15 mg/ml) was incubated with crude skeletal muscleextracts (2 mg/ml) in the presence of 0.25 mg/ml of ubiquitin at 37° C.for 0 or 20 minutes as described in Example 10. As shown in FIG. 7, theatrophying muscles dissected from the tumor-bearing rats exhibitedincreased ubiquitin conjugation to ¹²⁵I-α-lactalbumin

Northern blot analysis was carried out to measure the huE3αI and huE3αIImRNA expression in the gastrocnemius muscles of YAH-130 tumor-bearingmice. RNA from the dissected muscles was isolated with Trizol Reagent(Gibco, cat: 15596-018). The final RNA pellets were resuspended inDEPC-H₂O and 20 μg of total RNA per lane were separated byelectrophoresis through 1% agarose gels. The separated RNA wastransferred to nylon membranes and cross-linked to the filter byexposure to ultraviolet light.

The cDNA probes were generated by PCR with the following primers: forHu-E3α-I probe: 5′ primer, AGG AAG CTG TGG TCA TGT (SEQ ID NO: 22); 3′primer, GTT AGG AAG AAC AAC TG (SEQ ID NO: 23); for Hu-E3α-II probe: CTAAAG AAC AGC GAA GGC AAC AG (SEQ ID NO: 24); 3′ primer, CGC AGC TAC CCCAAC ACA TTA T (SEQ ID NO: 25). PCR was carried out for 30 cycles at 94°C. for 45 seconds, 50–58° C. for 45 seconds, and 72° C. for 1 minutesusing a commercially avaiblae kit (Boehringer Mannheim, cat: 1578553,).The PCR product was cloned into the pCR2.1 vector using the Original TACloning kit (Invitrogen). After digestion with EcoRI, the cloned PCRproduct was sequenced and confirmed.

The resulting cDNA probes were radiolabeled with [³²P]dCTP using thePrime-it RMT labeling kit (Stratagene, cat: 300392). Membranes wereprehybridized and hybridized (with the cDNA probes) in buffer containing1% BSA, 7% SDS, 0.5 M Sodium Phosphate (pH 7.0), 1 mM EDTA.Subsequently, the blots were washed in buffer containing 1% SDS, 0.04 MSodium Phosphate, 1 M EDTA, by the method of Church and Gilbert (Proc.Natl. Acad. Sci. U.S.A. 81:1991–1995, 1984) and exposed to radiographicfilm at −70° C. overnight.

As shown in FIG. 8, the expression of huE3αII mRNA was increased at day3 post-tumor implantation (see left panel) but the level of huE3αI hadnot changed significantly at day 3 (see right panel). This coincideswith the time point when the significant decrease in muscle mass wasdetectable in the C26 tumor-bearing mice (See Table VI in Example 12).The expression of both huE3αI and huE3αII was elevated at day 5post-tumor implantation in the tumor bearing rats. This corresponds to acachexia state with severe muscle wasting.

EXAMPLE 12 Expression of Human E3αI and Human E3αII in a Murine CancerCachexia Model of C26 Tumor Bearing Mice

The Colon-26 (C-26) tumor model of cachexia was used to demonstrate therole of huE3αI and huE3 αII as described in Matsumoto et al., Brit. J.Can. 79: 764–9 (1999) and Tanaka et al., Can. Res., 50: 2290–5 (1990).Seventy-two week old male CDF1 mice were injected in the left flank with0.2 ml containing either 0.5×10⁶ C26 cells or PBS. Following injection,body weight and food intake was observed daily. The pair fed controlmice (generated as described in Example 11) were fed the daily averagefood intake of the tumor bearing group. On the day 12 or 17post-injection, tumor bearing mice and pair fed control mice weresacrificed by CO₂ asphyxiation. Subsequently, a terminal serum samplewas collected and the kidney, heart and gastrocnemius muscles wererapidly dissected and weighed. The resulting C26 tumors were alsoweighed. The tissues were frozen on dry ice and stored at −70° C.

The wet weight of the skeletal muscles from the tumor-bearing mice weresignificantly less than the weight of those from the pair-fed controlmice as shown below in Table VI:

Days after Tumor Pair Fed Control Tumor-Bearing Percent Implantation n(in grams) (in grams) Change 12 days 12 0.127 ± 0.007 0.116 ± 0.0072−8.6% 17 days 12 0.117 ± 0.009 0.087 ± 0.001   −26% n = number ofanimals

RNA was isolated at day 12 and day 17 from the gastrocnemius muscle andcardiac muscle from the C26 tumor-bearing and pair-fed control mice asdescribed in Example 11. Northern blot analysis was carried out byloading 20 μg of total RNA per lane and seperating by electrophoresisthrough 1% agarose gels. The separated RNA was transferred to nylonmembranes and cross-linked to the filter by exposure to ultravioletlight.

The cDNA probes were generated by PCR with the following primers: formouse E3α-I probe: 5′ primer, TTT CTT CCA TTC CCT GCA TAC A (SEQ ID NO:26), 3′ primer, CAA AAC TTT ATA AAG GTG CCC GTA A (SEQ ID NO: 27), andfor Mouse E3α-II probe: 5′ primer, ATT CCC TGC ATG CAC TTC AGT AA (SEQID NO: 28), 3′ primer, CAT TCC CTG CAT GCA CTT CAG SEQ ID NO: 29). PCRwas carried out for 30 cycles at 94° C. for 45 seconds, 50–58° C. for 45seconds, and 72° C. for 1 minutes using a commercially available kit(Boehringer Mannheim cat: 1578553, ). The PCR product was cloned intothe pCR2.1 vector using the Original TA Cloning kit (Invitrogen). Afterdigestion with EcoRI, the cloned PCR product was sequenced andconfirmed.

The resulting cDNA probes were radiolabeled with [³²P]dCTP using thePrime-it RMT labeling kit (Stratagene, cat: 300392). Membranes wereprehybridized and hybridized (with the cDNA probes) in buffer containing1% BSA, 7% SDS, 0.5 M Sodium Phosphate (pH 7.0), 1 mM EDTA.Subsequently, the blots were washed in buffer containing 1% SDS, 0.04 MSodium Phosphate, 1 M EDTA, by the method of Church and Gilbert (Proc.Natl. Acad. Sci. U.S.A. 81:1991–1995, 1984) and exposed to radiographicfilm at −70° C. overnight.

As shown in FIG. 9, at day 12 after tumor-implantation tehrere was aclear increase in huE3αII mRNA expression in the skeletal muscles oftumor-bearing mice. Expression of both huE3αI and huE3 αII was increasedat day 17 post-implantation. Increased expression of huE3αII mRNAcoincides with the time point when the significant decease in musclemass became detectable in tumor-bearing mice (See Table VI above). Theexpression of huE3αI and huE3αII remained unchanged in the cardiacmuscle of the tumor-bearing mice. This corresponds to a cachexia statewith severe muscle wasting.

The data described in both Examples 11 and 12 show that in experimentalcachexia models, there was a sharp rise in the rate of ubiquitination inskeletal muscle tissues. The accelerated ubiquitination is due largelyto the activation of E3α, since addition of the E3α-selective inhibitor,arginine methylester, virtually abolished all the increasedubiquitination activities. In addition, the data demonstrated that intwo widely used experimental models of cachexia (murine C26tumor-bearing model and rat YAH-130 tumor-bearing model), the mRNAlevels of E3α-I and E3α-II increase significantly and specificallywithin skeletal muscle during the course of cachexia and muscle wasting.In these disease models and during the course of cachexia, the inductionof E3α-II occurred earlier than that of E3α-I and coincided with theearly onset of muscle wasting. During the late stage of cachexia, bothE3α-I and E3α-II were markedly induced when muscle wasting becamepronounced. Therefore, the results suggest that E3α-II may play a morecritical role in cachexia, although both E3α-I and E3α-II are apparentlyinvolved in the disease process.

EXAMPLE 13 Treatment of Muscle Cells with TNFα and IL-6 leads toIncreased Expression of Human E3αII and Increased Ubiquitination

Treatment with the proinflammatory cytokines, TNFα and IL-6, caused theinduction of huE3αII in C₂C₁₂ myotube cultures. C₂C₁₂ myoblasts werecultured in 100-mm dishes in an atmosphere of 5% CO₂ at 37° C. in DMEMsupplemented with 10% FBS and L-glutamine to reach 100% confluence.Myoblast differentiation was induced with DMEM supplemented with 2%horse serum and L-glutamine for 96 hours. Differentiated myotubes werethen treated with TNFα (10 ng/ml; R&D Systems cat no. 210-TA) or IL-6(10 ng/ml; R&D Systems cat no. 206-IL) for 3 days and 5 days.

After the 3 or 5 day incubation, RNA from differentiated C₂C₁₂ cultureswas isolated with Trizol Reagent and Northern blot analysis was carriedout as described in Example 11. Isolated RNA from untreated C₂C₁₂cultures were used as a control. The blots were hybridized with a³²P-labeled cDNA probes specific for muE3αI (lower panels) and muE3αII(upper panels). The probes were genreated as described in Example 12.

As shown in FIG. 10, the expression of muE3αII was markedly increased inthe cells treated with TNFα or IL-6 (See upper panels). Conversely, theexpression of muE3αI was not drastically induced in response toproinflammatory cytokine treatment. This data indicates a role for E3αIIin cytokine-mediated protein catabolism and muscle wasting.

Cytokine treatment also resulted in accelerated ubiquitination indifferentiated C₂C₁₂ cells. C₂C₁₂ cells were differentiated for 5 daysto allow formation of myotubes. The differentiated myotubes were treatedwith 2 ng/ml of IL-6 for 5, 24 or 48 hours. After the incubation, thecells were lysed and ¹²⁵I-Ubiquitin conjugation was carried out asdescribed in Example 10. As shown in FIG. 11, IL-6 treatment resulted ina marked increase in ubiquination of cellular proteins (left panel)which was detectable 5 hours post-treatment. The increase inubiquination was time dependent (see right panel).

Differentiated C₂C₁₂ myotubes were also treated with increasingconcentrations of TNFα (0, 3, 6, 10, 20 ng/ml) for one hour. Thistreatment resulted in a dose dependent increase in ¹²⁵I-ubiquitinconjugation of cellular proteins as shown in FIG. 12.

TNFα and IL-6 are major proinflammatory cytokines known to be involvedin cachexia and tissue wasting. The data reveals that these cytokinessignificantly upregulate the mRNA expression of E3αII in muscle cellsand stimulate muscle protein ubiquitination. Proinflammatory cytokines,such as TNFα, IL-6, IL-1, interferon-gamma, CNTF and leptin, have beenshown to be involved in disease states of cachexia and protein/tissuewasting, including cancer cachexia, renal cachexia (energy-proteinmalnutrition), burn cachexia and AIDS wasting. These findings that TNFαand IL-6 induce the expression of E3αII (FIG. 9) and stimulate proteinubiquitination in muscle cells (FIGS. 10 and 11) strongly suggest thatE3αII is critical target via which various cachectic factors induceprotein catabolism and cachexia/muscle wasting. This argument is furthersupported by our finding that recombinant expression of E3αII by cDNAtransfection leads to marked protein ubiquitination in myotube cultures(FIG. 5).

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

1. An isolated polypeptide comprising the amino acid sequence set forthin SEQ ID NO:
 2. 2. An isolated polypeptide comprising the amino acidsequence that is at least percent identical to the amino acid sequenceof SEQ ID NO: 2, wherein the polypeptide has E3α ubiquitin ligaseactivity of the polypeptide set forth in SEQ ID NO:
 2. 3. An isolatedpolypeptide comprising the amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as set forth in SEQ ID NO: 2with 1 to 100 conservative amino acid substitution(s), wherein thepolypeptide has E3α ubiquitin ligase activity of the polypeptide setforth in SEQ ID NO: 2; (b) the amino acid sequence as set forth in SEQID NO: 2 with 1 to 100 amino acid insertion(s), wherein the polypeptidehas E3α ubiquitin ligase activity of the polypeptide set forth in SEQ IDNO: 2; (c) the amino acid sequence as set forth in SEQ ID NO: 2 with 1to 100 amino acid deletion(s), wherein the polypeptide has an E3αubiquitin ligase activity of the polypeptide set forth in SEQ ID NO: 2;(d) the amino acid sequence as set forth in SEQ ID NO: 2 which has a C-and/or N-terminal truncation up to about 100 amino acids, wherein thepolypeptide has E3α ubiquitin ligase activity of the polypeptide setforth in SEQ ID NO: 2; and (e) the amino acid sequence as set forth inSEQ ID NO: 2, with a modification of 1 to 100 amino acids consisting ofamino acid substitutions, amino acid insertions, amino acid deletions,C-terminal truncation, and N-terminal truncation, wherein thepolypeptide has E3α ubiquitin ligase activity of the polypeptide setforth in SEQ ID NO:
 2. 4. An isolated polypeptide encoded by the nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of: (a) the nucleotide sequence as set forth in SEQ ID NO: 1;(b) a nucleotide sequence encoding the polypeptide set forth in SEQ IDNO: 2; (c) a nucleotide sequence which hybridizes under highly stringentconditions to the complement of the coding sequence of (a) or (b),wherein said stringent conditions comprise a final wash with 0.015 Msodium chloride and 0.0015 M sodium citrate at 65–68° C. in 0.1×SSC and0.1% SDS or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50%formamide at 42° C., wherein the nucleotide sequence encodes apolypeptide which has E3α ubiquitin ligase activity of the polypeptideset forth in SEQ ID NO:2; and (d) a nucleotide sequence fullycomplementary to any of (a)–(c) over the full length of the sequence. 5.The isolated polypeptide according to claim 2 wherein the percentidentity is determined using a computer program selected from the groupconsisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, andthe Smith-Waterman algorithm.
 6. A composition comprising thepolypeptide of claim 1, 2, 3, or 4 and a pharmaceutically acceptableformulation agent.
 7. The composition of claim 6 wherein thepharmaceutically acceptable formulation agent is a carrier, adjuvant,solubilizer, stabilizer, or anti-oxidant.
 8. The composition of claim 6wherein the polypeptide comprises the amino acid sequence as set forthin SEQ ID NO:
 2. 9. A chemically modified derivative of the polypeptideof claim 1, 2, 3, or
 4. 10. The polypeptide derivative of claim 9 whichis covalently modified with a water-soluble polymer.
 11. The polypeptideof claim 10 wherein the water-soluble polymer is selected from the groupconsisting of polyethylene glycol, monomethoxy-polyethylene glycol,dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol,propylene glycol homopolymers, polypropylene oxide/ethylene oxideco-polymers, polyoxyethylated polyols, and polyvinyl alcohol.
 12. Afusion polypeptide comprising the polypeptide of claim 1, 2, 3, or 4fused to a heterologous amino acid sequence.
 13. The fusion polypeptideof claim 12 wherein the heterologous amino acid sequence is an IgGconstant domain or fragment thereof.
 14. A process of producing a humanE3α ubiquitin ligase polypeptide comprising: a.) inserting an isolatednucleic acid molecule encoding a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 2 into a vector; b.) inserting saidvector into a host cell; c.) culturing said host cell under suitableconditions to express the polypeptide; and d.) optionally isolating thepolypeptide from the cultured host cell.
 15. A method of identifying acompound which binds to a polypeptide comprising: (a) contacting thepolypeptide of claim 1, 2, 3, or 4 with a compound; and (b) determiningthe extent of binding of the polypeptide to the compound.